Method of using fusion proteins

ABSTRACT

The present invention concerns a family of nucleic acids, polypeptides and cloning vectors which direct expression of fusion proteins that can mimic aggregated IgG (AIG) and immune complex function with respect to their interactions with FcγR and which allow for the inclusion and targeting of a second protein domain to cells expressing FcγR. This was accomplished by expressing multiple linear copies of the hinge and CH2 domains (HCH2) of human IgG 1  fused to the framework region of human IgG 1 . Convenient restriction sites allow for the facile introduction of additional amino-terminal domains. Methods for treating patients using fusion proteins are also disclosed. The HCH2 polymers described here represent a new strategy in the design of recombinant proteins for the therapeutic targeting of FcγR in autoimmune disorders.

This application is a divisional of U.S. application Ser. No. 12/212,776(now U.S. Pat. No. 7,897,729), filed Sep. 18, 2008, which isincorporated by reference in its entirety, which is a continuation ofU.S. application Ser. No. 10/096,521 (now U.S. Pat. No. 7,511,121),filed Mar. 11, 2002, which is incorporated by reference in its entirety,which claims benefit to U.S. Provisional Application No. 60/274,392,filed Mar. 9, 2001, which is incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

A. Field of the Invention

The present invention relates generally to the field of immunology. Morespecifically, the present invention relates to immune complexes and theexpression and use of recombinant proteins containing multiple hinge andCH2 regions of immunoglobulins.

B. Description of Related Art

Immune complexes (IC) exhibit diverse biological activities; some thatcontribute to disease whereas others ameliorate disease. Deposition ofIgG containing IC on tissue surfaces, as for example in glomeruli, cancontribute to the pathogenesis of antibody-mediated autoimmune diseases.On the other hand, IC can favorably modulate T- and B-cell activationpathways via binding to Fc receptors expressed on immunocytes.Aggregated IgG (AIG) shares many features and biological activities withIC. Both modulate T-cell suppressor function (Antel of al., 1981;Durandy et al., 1981), cytokine synthesis, IgG secretion, and lymphocyteproliferation (Berger et al., 1997; Wiesenhutter et al., 1984; Ptak etal., 2000).

Monomeric IgG, or the Fc fragment thereof, ameliorates diseaseprogression in animal models of autoimmune disease (Miyagi et al., 1997;Gomez-Guerrero et al., 2000). Monomeric IgG is widely usedtherapeutically, usually in massive doses, to treat antibody-mediateddiseases in man. The protective effect in antibody-mediated diseases maybe achieved in part through blockade of FcγRs such that binding of IC tothem is impeded (Clynes et al., 1998). IgG administration also favorablyaffects the course of T-cell mediated autoimmune diseases such asmultiple sclerosis (Fazekas et al. 1997; Sorensen et al., 1998; Achironet al., 1998). Here the basis for benefit is poorly understood though itis postulated to involve the increased production of anti-inflammatorycytokines initiated by binding of IV IgG, or complexes derivedtherefrom, to FcγR. In both antibody and T-cell mediated processes themechanisms and consequences of FcγR engagement are fundamental to theunderstanding and treatment of autoimmune diseases.

Aggregated IgG has been proposed as a treatment for autoimmune diseasesof humans. The use of aggregated IgG has been studied as a treatment formultiple sclerosis and other autoimmune diseases. However, aggregatedIgG has major limitations. IgG is commonly aggregated by exposure toheat; the resultant aggregates are bound together in a random fashionlimiting reproducibility from one preparation to the next. Preparationscontain a heterogeneous collection of aggregates of varying size indiverse conformations.

The formation of immunoglobulin fusion proteins is known in the art. Forexample, U.S. Pat. Nos. 5,714,147 and 5,455,165 disclose novel hybridimmunoglobulin molecules and the expression vectors encoding them. Thesechimeric molecules are used in improving the circulating plasmahalf-life of ligand binding molecules, and comprise a lymphocyte homingreceptor fused to an immunoglobulin constant region. Homo orhetero-dimers or tetramer hybrid immunoglobulins containingpredominantly the heavy and light constant regions of immunoglobulin areused. U.S. Pat. No. 6,046,310 discloses FAS ligand fusion proteinscomprising a polylpeptide capable of specifically binding an antigen orcell surface marker for use in treatment of autoimmune disorders. Thefusion protein preferably comprises IgG2 or IgG4 isotype, and maycomprise antibodies with one or more domains, such as the CH2, CH1 orhinge deleted. Majeau et al. (1994) discusses Ig fusion proteins usedfor the inhibition of T cell responses. These fusion proteins compriseIgG1 and LFA-3. Eilat et al. (1992) disclose a soluble chimeric Igheterodimer produced by fusing TCR chains to the hinge region, CH2, andCH3 domains of human IgG1.

Immunoglobulin fusion proteins have been employed to express numerousproteins in mammalian and insect cells (Ashkenazi, et al., 1997). Fusionprotein platforms can permit the introduction of additional functions,for example, inclusion of the amino-terminal CD8α domain may result inthe co-ligation of FcR on lymphocytes to MHC I on antigen presentingcells (Alcover, et al., 1993; Meyerson, et al., 1996).

Other Ig proteins and variants have also been studied for theirtherapeutic effect on autoimmune diseases, including a recombinantpolymeric IgG that mimics the complement activity of IgM (Smith andMorrison, 1994) where the polymeric IgG is formed by the polymerizationof H₂L₂ subunits. Greenwood et al. (1993) discusses therapeutic potencyrelative to the structural motifs involving the human IgG antibodies,IgG1, IgG3, and IgG4. U.S. Pat. No. 5,998,166 discloses human FcγR-IIIvariants, which can be used in the therapy and/or diagnosis ofautoimmune diseases. U.S. Pat. No. 5,830,731 discloses novel expressionvectors in which cell surface antigens cloned according to thatinvention have diagnostic and therapeutic utility in immune-mediatedinfections. Cell surface antigens that are used to regulate lymphocyteactivation, achieve antigen aggregation in vitro by incubatinglymphocytes with immobilized ligands or antibodies or their fragments(WO9942077). However, the aggregated IgG and Fc aggregates have limitedreproducibility, containing a random and heterogeneous mixture ofprotein thereby limiting their effectiveness as therapeutic agents.Other problems include a lack of an ability to target a number of celltypes with a single agent and size limitations.

SUMMARY OF THE INVENTION

The present invention provides novel polypeptides that contain varyingnumbers of FcγR and/or complement binding domains. These polypeptidesprovide several advantages over other FcγR-binding moieties, includingIgG, Fc fragments, IgG fusion proteins, and complexes and/or aggregatesthereof. These advantages include: the ability to hone interaction withFcγR's by varying the construct design and protein product and theability to obtain a precisely defined construct and protein productcontaining a known number of FcγR binding domains as opposed to theheterogeneous nature and considerable variations between batches whenusing aggregated IgG (AIG); an increase in the potency compared to AIG;and the ability to target a number of cell types (e.g. NK cells,monocytes, and B cells) with a single agent. Therefore, the inventionallows one to design polypeptides that contain multiple FcγR bindingsites and mimic aggregated proteins that are capable of moderatingdisease severity. Using the teachings of this specification according tothe invention, one of ordinary skill is able to create a homogeneousprotein with a small size range and conformation. One is also able tocreate soluble fusion proteins to facilitate generation of dose responsecurves over a range of concentrations.

Some embodiments of the invention relate to polypeptides comprising afirst region comprising a protein or portion thereof and a second regioncomprising more than one copy of at least a portion of an HCH2 region ofan IgG. These polypeptides may target to cells expressing FcγR, bind toFcγR, and/or bind complement components. These polypeptides may furthercomprise an immunoglobulin framework region. In some aspects of theinvention, the polypeptides do not comprise an IgG constant region. Insome preferred embodiments, the polypeptides may be single chains,dimers, or trimers, when in active form. The preferred size of thepolypeptide is between 26 kDa and 1500 kDa or more preferably between 45kDa and 600 kDa. It is an aspect of the current invention that thepolypeptide is soluble in aqueous solution.

Some aspects of the invention comprise adapting the polypeptide to mimicaggregated IgG and immune complex functions in interactions with FcγR.The amino acid sequence is preferably of a mammalian immunoglobulin. Insome cases, the polypeptide may comprise at least two immunoglobulinsequences. The immunoglobulin may be of any source, including, but notlimited to a human, rodent, cow, goat, sheep, horse, dog, cat, or pig.The immunoglobulin is more preferably a murine or human immunoglobulin.More specifically, the polypeptide may comprise an amino acid sequenceof human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM, and, in somecases an amino acid sequence comprising a sequence of at least two ofhuman IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, and IgM. In some cases thepolypeptide is adapted to target to cells expressing one or more ofFcγR, FcαR, FcεR, FcμR, FcδR, or FcRn under appropriate conditions. Insome preferred embodiments, the polypeptide is adapted to bind to atleast two of FcγR, FcαR, FcεR, FcμR, FcδR, or FcRn.

In some preferred embodiments the first region of the polypeptidecomprises at least a portion that comprises a binding site for a moietyin an organism or on a cell. For example, the first region may comprisea sequence from a Fab of an antibody or antibody-like protein, a CD8α,human serum albumin (HSA), or a transporter protein. In some preferredcases where the first region comprises a sequence from a Fab, the Fab isfrom a human antibody or a humanized antibody. In some preferredembodiments, the moiety in the organism or on the cell is a cell surfacemarker, with the cell surface marker, in some cases being an antigen.For example, the sequence may bind to a tumor-associated antigen or anantigen indicative of viral infection. In other cases, the binding maybe a least a portion of a binding site of a cellular receptor, areceptor ligand, or an adhesion molecule. In some preferred embodiments,the first region of the polypeptide comprises binding sites that bindtwo separate antigens, or cell surface markers.

The second region of the polypeptide may comprise any number of copiesof at least a portion of the HCH2 region, including, but not limited to,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, and/or 25 or morecopies. Further, the invention contemplates any range derivable betweenany of the above-described integers. An aspect of the current inventioncomprises making amino acid substitutions that will alter the tendencyof the polypeptide to aggregate and/or an altered hinge region, whichpreserves FcγR and/or complement binding and prevents inter-moleculardisulfide bond formation. These substitutions may be made in any mannerknown to those of skill in the art and described herein.

In some preferred embodiments, the HCH2 region is an HCH2 region fromhuman IgG1. Further, in some preferred embodiments, the HCH2 regioncomprises at least amino acid residues 233 to 239 of human IgG1 heavychain (Eu numbering). In some other embodiments, the HCH2 regioncomprises at least amino acid residues 216 to 340 of human IgG1 heavychain (Eu numbering), which residues include the entirety of the humanIgG1 hinge and CH2 portions. However, as one of ordinary skill in theart will understand in view of the disclosures herein, there are manydifferent precise embodiments of the claimed polypeptides that may beprepared and tested according to the teaching of the invention. It ispossible for the individual HCH2 regions to comprise any number ofresidues selected from the hinge and CH2 regions of an IgG, so long asthe requisite activity for the polypeptide is obtained. Therefore, theinvention encompasses, and this specification describes, polypeptideswherein the HCH2 regions comprise any one contiguous portion orcombination of contiguous portions of any HCH2 of any type, whichportions or contiguous portions may be mutated or modified away from anative HCH2 sequence in any manner possible, so long as the polypeptideshave an activity as described herein.

A further aspect of the current invention comprises amino acidsubstitutions in the second region of the polypeptide that will increasethe specific binding of the polypeptide for certain subtypes of FcγRand/or decrease the specific binding of the polypeptide for othersubtypes of FcγR. Those of skill in the art will be able to prepare,test and use polypeptides comprising such mutations by following theteaching of this description.

It is another aspect of the current invention that a linker operablylinks the first and second regions of the polypeptide. This linker canbe any form of molecule that links the first and second regions byeither covalent or non-covalent forces. In particular, the linker can bea peptide or polypeptide of any length that will function in the contextof the invention, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 40, 50, or more amino acids, or any number or range of amino acidsthat will work for the purposes of the invention. Those of ordinaryskill in the art will be able to determine and test any number of typesand lengths of linkers.

It is an aspect of the current invention that the polypeptide is adaptedto treat immune deficiency disorder, dermatomyositis-polymyositis, aninfectious disease, autoimmune thyroiditis, interstitial cystitis,prostatitis, an inflammatory disease, an autoimmune disease, an allergicdisease, a degenerative disease of the central nervous system, a diseaseof the platelets, a disease of the blood vessels, inflammatoryneuropathy, a traumatic condition, rheumatoid arthritis, lupus, and/orasthma. In preferred embodiments, the polypeptide is adapted to treat aninflammatory disease of the central nervous system such as multiplesclerosis, an inflammatory bowel disease such as Crohn's Disease orulcerative colitis, autoimmune diseases of the eye such as uveitis orretinitis, an allergic disease such as allergic rhinitis, a degenerativedisease of the central nervous system such as Alzheimer's disease or ALS(Lou Gehrig's disease), a disease of the white blood cells such as alymphoma or a leukemia, a disease of platelets such as ImmuneThrombocytopenic Purpura, a disease of the blood vessels such asKawasaki disease or atheroma, an inflammatory neuropathy such asGuillain Barré Syndrome, and/or a traumatic condition such as a spinalcord injury. The polypeptide may also be adapted to promote apoptosis,necrosis, or lysis or decrease cell division of neoplastic or virallyinfected cells, or to alter complement binding.

It is a further aspect of the current invention that the polypeptide cantarget NK cells or that the polypeptide can target NK cells, monocytes,macrophages, dendritic cells, T cells or B cells. Further, thepolypeptide may be adapted to induce immune tolerance. The polypeptidemay, likewise, be adapted for use as a vaccine or for use as anantigen-presenting vehicle.

Further embodiments of the invention relate to nucleic acids that encodepolypeptides according to the invention. These nucleic acids, in someembodiments, encode both the first region comprising a protein orportion thereof and the second region comprising more than one copy ofat least a portion of an HCH2 region of an IgG. In other embodiments,the nucleic acids encode only one of the first polypeptide regioncomprising a protein or portion thereof and the second region comprisingmore than one copy of at least a portion of an HCH2 region of an IgG.Further, those of ordinary skill will understand that some nucleic acidsaccording to the invention may encode only portions of either of theseregions, and will be able to employ such nucleic acids.

In some preferred aspects of the invention, the nucleic acids areexpressible. The polypeptides may be expressed in prokaryotic cells oreukaryotic cells or expressed in a cell free system. Preferred cells forexpression include, but are not limited to, insect cells and mammaliancells. Preferred mammalian cells include human and rodent cellsincluding murine and CHO cells. The nucleic acid may be expressible as atransgene or expressible in a genetically modified animal, for example amouse. The polypeptide may be further defined as an immunoglobulinfusion protein.

In certain preferred embodiments, the nucleic acid allows for expressionof a polypeptide comprising the first region and the second region as asingle active fusion polypeptide, without the need for any additionalprotein or other components for function. In the embodiments, thenucleic acid will allow for the expression of either of the first regionor the second region, or a portion of either region. In cases where onlya single region of the polypeptide, or portion thereof, is encoded bythe nucleic acid, then those of ordinary skill will know how to expressthose portions and, if they so desire, assemble and/or process them intofully functional polypeptides according to the invention. Such assemblyand/or processing can employ any of the methods described herein orknown to those of skill and/or any of the linkers described herein orknown to those of skill.

Another aspect of the current invention comprises vectors that comprisea nucleic acid encoding all or part of a polypeptide of the presentinvention. The vectors may, for example, be cloning or expressionvectors. In some cases, these vectors are produced by a method whereinthe nucleic acid sequence encoding the second region comprising morethan one copy of at least a portion of an HCH2 region of an IgG isinserted into an existing antibody sequence including a monoclonalantibody sequence or the sequence of any expressible protein.

The cloning vectors of the invention may be comprised in any suitablerecombinant host cell, as described elsewhere herein or known to thoseof skill in the art.

In other aspects, the invention relates to methods comprising: obtaininga polypeptide according to the invention, i.e., a polypeptide comprisinga first region comprising a protein or portion thereof and a secondregion comprising more than one copy of at least a portion of an HCH2region of an IgG; and administering the polypeptide to a cell. In somesuch methods, the protein or portion thereof is specifically defined ascomprising a soluble protein domain, a transporter domain and/or aligand-binding domain. Additionally, the polypeptide further comprisesan immunoglobulin framework region.

The methods of the invention are further defined, in some cases, asmethods of treating an organism with the polypeptides of the invention.In many preferred embodiments, the organism is a mammal, for example,but not limited to, a human, rodent, horse, dog, cat, pig, cow, or goat.In some particularly preferred embodiments, the mammal is a human inneed of treatment for a disease, condition, or disorder. In terms ofrodents, some particularly preferred rodents are mice and rats.

In the methods of the invention the treating of an organism may occur inany manner, including, but not limited to oral treatment, intranasaltreatment, or injection. For example, injection may include, but not belimited to, intravenous, intraperitoneal, intramuscular, or subcutaneousinjection. Of course, those of skill will understand that there are manyroutes of treatment possible, and are enabled by this specification toperform them.

The methods of treating an organism will involve treatment with anamount of the polypeptide that is effective to treat the disease,condition, or disorder that the organism has, or is suspected of having,or to bring about a desired physiological effect. In many cases, thisamount will be less than the amount of an IgG or aggregated IgG proteinused to treat a comparable disease, condition, or disorder or to bringabout a comparable desired physiological effect. In some preferredembodiments, the amount of the polypeptide is administered at aconcentration of 0.05 to 10 mg/kg body weight, or more preferably 0.2 to5 mg/kg body weight, or even more preferably 0.5 to 2 mg/kg body weightor 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1.0 mg/kg, 1.2mg/kg, 1.4 mg/kg, 1.6 mg/kg, 1.8 mg/kg or 2.0 mg/kg. In regard to someconditions, it is preferred that the dosage will be about 0.75 mg/kgbody weight. Of course, those of skill in the art will appreciate thatit is possible to employ many concentrations in the methods of thepresent invention, and will be able to adjust and test any number ofconcentrations in order to find one that achieves the desired result ina given circumstance. For example, if the polypeptides of the inventionare administered in combination with one or more other therapeutic agentfor a given disease, condition, or disorder, it is possible to achieve asynergistic effect between the other therapeutic agent and thepolypeptide, such that less of the polypeptide is needed.

In some specific embodiments, the polypeptides of the invention are usedto treat a mammal that has immune deficiency disorder,dermatomyositis-polymyositis, an infectious disease, autoimmunethyroiditis, interstitial cystitis, prostatitis, an inflammatorydisease, any autoimmune disease, an allergic disease, a degenerativedisease of the central nervous system, a disease of the platelets, adisease of the blood vessels, inflammatory neuropathy, a traumaticcondition, rheumatoid arthritis, lupus, and/or asthma. In preferredembodiments, the polypeptide is used to treat an inflammatory disease ofthe central nervous system such as multiple sclerosis, an inflammatorybowel disease such as Crohn's Disease or ulcerative colitis, autoimmunediseases of the eye such as uveitis or retinitis, an allergic diseasesuch as allergic rhinitis, a degenerative disease of the central nervoussystem such as Alzheimer's disease or ALS (Lou Gehrig's disease), adisease of the white blood cells such as a lymphoma or a leukemia, adisease of platelets such as Immune Thrombocytopenic Purpura, a diseaseof the blood vessels such as Kawasaki disease or atheroma, aninflammatory neuropathy such as Guillain Barré Syndrome, and/or atraumatic condition such as a spinal cord injury. The polypeptide mayalso be used to promote apoptosis, necrosis, or lysis or decrease celldivision of neoplastic or virally infected cells, or to alter complementbinding.

In some aspects of the invention, the methods of the invention arefurther defined as methods of altering immunity in a mammal comprisingadministering a polypeptide of the invention to the mammal.

The invention also relates to methods of killing neoplastic cellscomprising treating a neoplastic cell with one or more polypeptides ofthe invention. In many such cases, the neoplastic cell is a carcinomacell, tumor, and/or other form of cancer cell. In some preferredembodiments, the treating results in complement-dependent cytotoxicity,antibody-dependent cell-mediated cytotoxicity, or complement-dependentcell-mediated cytotoxicity of the neoplastic cell. In many cases, theneoplastic cell is comprised in an organism, such as, but not limitedto, any of the organisms described above and elsewhere in thisspecification. In some particularly preferred embodiments, the inventionrelates to methods of using the polypeptides of the invention asdescribed herein to treat a human who has cancer. In other cases, theneoplastic cell is in cell culture.

The methods of the invention may be further defined as a method ofkilling a virally infected cell comprising treating a virally infectedcell with a polypeptide of the invention. In some preferred such cases,the treatment results in complement-dependent cytotoxicity,antibody-dependent cell-mediated cytotoxicity, or complement-dependentcell-mediated cytotoxicity of the virally infected cell. In someembodiments, the virally infected cell is comprised in a mammal, forexample, a human or rodent.

While many of the more commercially valuable aspects of the methods ofthe invention relate to methods of treating organisms, especiallyhumans, who have some form of disease, disorder or condition, there areother reasons for treating animals with the polypeptides of theinvention. For example, it is possible to treat a laboratory animal withthe polypeptides in order to test whether or not the polypeptides willbe useful in treating humans or other animals in a real-life situation.

In other aspects of the methods of the invention one is able to use thepolypeptides of the invention to treat cells in cell culture. There isany number of reasons known to those of skill in the art for wanting totreat cells in culture with the polypeptides. For example, one may wantto test polypeptides produced according to the methods described hereinfor any utility for the given specific methods described herein. Also,one may wish to use the polypeptides of the invention to kill neoplasticor virally infected cells in culture or a assay immune cell function invitro.

The invention also relates to methods of delivering a therapeutic agentto a delivery site in a mammal comprising: providing a polypeptidecomprising a first region which targets the delivery site and a secondregion comprising more than one copy of at least a portion of an HCH2region of an IgG; and providing the therapeutic agent to the mammal;wherein the therapeutic agent is delivered to the delivery site to treatthe mammal. The polypeptides in this aspect of the invention may furthercomprise an immunoglobulin framework region. The therapeutic agent canbe any form of therapeutic agent known to those of skill in the art. Forexample, the thereaeutic agent, may be a vaccine component, monoclonalantibody, cytokine, interleukin, steroid, interferon, toxin,chemotherapeutic agent, radioisotope, or immunomodulatory agent such asglatiramer acetate and interferon-β. Further, the invention relates tomethods of delivering labels to sites in mammals for imaging or otherrelated procedures. These labels may be any form of labeling moietyknown to those of skill in the art, including, but not limited to,fluorescent labels, affinity labels, radiolabels, etc.

Further, the invention relates to methods of preparing one or more of animmunological product comprising: immunizing a mammal with an amount ofan antigen and a polypeptide comprising a first region comprising morethan one copy of at least a portion of an HCH2 region of an IgG; andproducing an immunological product in the mammal. In these embodimentsof the invention, the polypeptide may be any of the polypeptidesdescribed above. Further, the immunological product may comprises atleast one T cell, product produced by a T cell, B cell, or antibodyproduced by a B cell, wherein the product is directed against theantigen. In some cases, the method is a method of obtaining animmunological product for further use, and the immunological product isobtained from the mammal. In other cases, the method is a method ofvaccinating or inducing immunity against the antigen in the mammal. Insome aspects of these methods of the invention, the first regioncomprises a portion of or all of an antibody. Further, the first regionmay be adapted to bind to tumor, a virus, a fungus, a rickettsia, amycoplasma, a bacterium, a protozoal parasite or a metazoal parasite.Additionally, the first region may be conjugated to a protein ornon-protein molecule derived from a tumor, a virus, a fungus, arickettsia, a mycoplasma, a bacterium, a protozoal parasite, or ametazoal parasite.

In this specification and the claims, the words “a” and “an,” when usedwith the conjunction “comprising,” mean “at least one” or “one or more.”

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1—Schematic depicts the design rationale utilized in theconstruction of the HCH2 polymer, the key feature of which is theiterative regeneration of cloning sites in the extension step. The ΔHCH2shown in the schematic represents an HCH2 monomer in which the hingecysteines have been changed to serines.

FIG. 2—Schematic representing fusion protein constructs. The trianglerepresents the leader sequence and amino-terminal domain which iscomprised of either the first 119 amino acid residues from theextracellular domain of human CD8α or domain I of HSA. The boxes,labeled HCH2, represent repeat units comprised of the hinge and CH2domains of human IgG1 (amino acid residues 226-350). The element labeledFRM represents the framework region comprised of the hinge, CH2 and CH3domains of human IgG1 (amino acid residues 226-457). RO, indicatesframework without repeat units, R2, framework with 2 repeat units, R3,framework with 3 repeat units, R4, framework with 4 repeat units.

FIGS. 3A, and 3B, and 3C.—Western Blot analyses. FIG. 3A. Recombinantproteins were separated on 7% SDS-PAGE gels and stained with Coomassiebrilliant blue dye to reveal protein. FIG. 3B. Recombinant proteins weretransferred to nitrocellulose membrane and stained with antibodiesdirected against human Fc. Note that the human IgG control and thefusion proteins are recognized by anti-Fc antibody. FIG. 3C. Recombinantproteins were transferred to nitrocellulose membrane and stained withantibodies directed against human CD8α. Note that only the fusionproteins are recognized by anti-CD8α antibody, indicating specificdetection of CD8α.

FIG. 4—HSAR4, immobilized anti-CD16 mAb 3G8, and aggregated IgG induceproliferative responses from PBMC in a dose-dependent and IL2-dependentmanner. 2×10⁵ freshly isolated PBMC were plated into 96 well plates inthe presence of IL-2 (1 ng/mL) and immobilized anti-CD16 mAb 3G8together, IL-2 (1 ng/mL) and HSAR4 together, or IL-2 (1 ng/mL) and AIGtogether for 72 hr. During the last 5 hr the cells were pulsed with 1μCi of [methyl-³H] thymidine. The graph compares the proliferativeresponse of PBMC to varying dilutions of each reagent. CPM is shown onthe y-axis and micrograms/ml of stimulus used is shown on the x-axis.Proliferative responses in the presence of medium alone were 787±447 andin the presence of IL-2 alone were 1957±1117. Data represent the averagefrom four individuals±SEM.

FIG. 5—PBMC activation with HCH2 polymer proteins correlates with thenumber of HCH2 region repeats indicating a high level of sensitivity ofFcγ receptors to HCH2 number in the HCH2 polymer proteins. 2×10⁵ freshlyisolated PBMC were plated into 96 well plates in the presence of mediumalone, or with IL-2 (1 ng/mL) and varying concentrations of HSAR0,HSAR2, HSAR3, or HSAR4 for 72 hr. During the last 5 hr the cells werepulsed with 1 μCi of [methyl-³H] thymidine. The graph compares theproliferative response of PBMC to varying dilutions of each HCH2 polymerprotein used. CPM is shown on the y-axis and micrograms/ml of HCH2polymer protein used is shown on the x-axis. Proliferative responses inthe presence of medium alone were 803±1069 and in the presence of IL-2alone were 2903±962. Proliferative responses in the presence of HSAR0,HSAR2, HSAR3, and HSAR4 in the absence of IL-2 were 1027±176, 1531±504,1237±379, and 1661±592 respectively. Data represent the average fromfour individuals+SEM.

FIG. 6—C1q binding assay. Human IgG, HSAR0, or HSAR4 were allowed tobind overnight to 96-well plates at a concentration of 1 μg/mL. The nextday the plates were washed and the immobilized proteins were incubatedwith human C1q at the indicated concentrations for 4 hours in PTGbuffer. Bound C1q was detected with goat anti-human C1q polyclonalantibodies. The results demonstrate that HCH2 polymers expressed ininsect cells engage C1q weakly.

FIG. 7—EAE was induced in SJL/J mice with PLP peptide in completeFreund's adjuvant. Mice were treated with HSAR4 (50 μg/0.150 ml saline,given i.p.), HSAR0 (50 μg/0.150 ml saline, given i.p.) or saline alonethree days prior to, 1 day after, and 3 days after, immunization withPLP peptide. Clinical disease was graded on a scale of 0 to 5 ofincreasing severity; 0, no abnormality; 1, flaccid tail; 2, flaccid tailwith mild hind limb weakness; 2.5, moderate hind leg weakness but notcomplete paralysis; 3, total paralysis of hind legs, 4, hind legparalysis with forelimb weakness or paralysis; 5, moribund. Mice thatbecame moribund were sacrificed. FIG. 7 compares disease scores of micetreated with saline alone to those treated with HSAR4 or HSAR0. Miceinjected with HSAR4 displayed less severe acute disease than miceinjected with saline alone or with HSAR0.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

This invention describes a family of nucleic acids with a regionencoding a polypeptide and the polypeptide encoded by the nucleic acidwhich can mimic aggregated IgG (AIG) and immune complex function withrespect to their interactions with FcγR and which allow for theinclusion and targeting of a second protein domain to cells expressingFcγR. The invention also describes a family of cloning vectors whichdirect expression of fusion proteins and fusion proteins that can mimicaggregated IgG (AIG) and immune complex function with respect to theirinteractions with FcγR and which allow for the inclusion and targetingof a second protein domain to cells expressing FcγR. Expressing multiplelinear copies of at least portions of the hinge and CH2 domains (HCH2)of human IgG₁ fused to the framework region of human IgG₁ gives arecombinant protein with these features. Convenient restriction sitesallow for the facile introduction of additional amino-terminal domains.The resulting molecule is tripartite. The carboxyl-IgG₁ framework domainprovides stability and permits dimerization; the intervening HCH2polymer confers increased effector function, including targeting tosubsets of cells expressing FcR, increased capacity to ligate FcR and/orto bind complement components, while the amino terminal domain candeliver an additional signal to cells expressing FcγR.

Another aspect of the invention describes methods for preparing thepolypeptides and fusion proteins of the invention as well as methods forthe use of the polypeptides and fusion proteins of the invention.Methods include treating inflammatory diseases, altering immunity,killing neoplastic cells, and delivering therapeutic agents to adelivery site.

A. ANTIBODY STRUCTURE

Antibodies comprise a large family of glycoproteins with commonstructural features. An antibody is comprised of four polypeptides thatform a three dimensional structure which resembles the letter Y.Typically, an antibody is comprised of two different polypeptides, theheavy chain and the light chain.

An antibody molecule typically consists of three functional domains: theFc, Fab, and antigen-binding site. The Fc domain is located at the baseof the Y. The arms of the Y comprise the Fab domains. Theantigen-binding site is located at the end of each arm of the Y. Thearea at the fulcrum of the arms of the Y is the hinge region.

There are five different types of heavy chain polypeptides designated asα, δ, ε, γ, and μ. There are two different types of light chainpolypeptides designated κ and λ. An antibody typically contains only onetype of heavy chain and only one type of light chain, although any lightchain can associate with any heavy chain.

Antibody molecules are categorized into five classes, IgG, IgM, IgA, IgEand IgD. The IgG class is further divided into subclasses includingIgG1, IgG2, IgG3 and IgG4 for human IgG. An antibody molecule iscomprised of one or more Y-units, each Y comprising two heavy chains andtwo light chains. For example IgG consists of a single Y-unit IgM iscomprised of 5 Y-like units.

The carboxyl terminal of each heavy chain polypeptide is known as theconstant (Fc) region. The amino terminal of each heavy and light chainpolypeptide is known as the variable (V) region. Within the variableregions of the chains are hypervariable regions known as complementaritydetermining regions (CDRs). The variable regions of one heavy chain andone light chain associate to form an antigen-binding site. Each heavychain and each light chain includes three CDRs. The six CDRs of anantigen-binding site define the amino acid residues that form the actualbinding site for the antigen. CDR variability accounts for the diversityof antigen recognition.

The mature human IgG1 heavy (H) chain typically spans 447 amino acidresidues. The Fc region of the H chain is essentially the same for allIgG1 heavy chain molecules. The Fe region is the portion of the IgG1polypeptide that interacts with Fc receptors. The Fc region can befurther subdivided into three consecutive regions, the hinge, the CH2,and the CH3 domains. The binding site for Fc receptors is found withinthe hinge and CH2 (HCH2) domains of human IgG1. The HCH2 regionencompasses amino acid residues 216 to 340 of the human IgG1 H chain (Eunumbering). The hinge region spans residues 216 to 237 whereas the CH2domain encompasses residues 238 to 340.

B. IMMUNOGLOBULIN FUSION PROTEINS

Recombinant immunoglobulin fusion proteins are well known in the art.For example, see Capon, et al., 1989; Traunecker, et al., 1989; Chamow,et al., 1996; Ashkenazi, et al., 1997. Typically, a recombinantimmunoglobulin fusion protein has an amino-terminus composed of aligand-binding domain fused to a carboxyl-terminus composed of thehinge, C_(H)2, and C_(H)3 regions of Ig. The Ig class most commonly usedis IgG1. The hinge, C_(H)2, and C_(H)3 regions of IgG are collectivelyreferred to as the Fc region of IgG. The hinge region provides aflexible linker between the Fc region and the ligand binding domain. Italso is the site of inter-chain disulphide bond formation, ie., thecovalent linking of one antibody chain to another to make the familiardimeric structure. The hinge region (especially the part nearest to theCH2 domain known as the hinge proximal region) is necessary formolecular recognition and binding to Fcγ receptors and complementcomponents. Thus recombinant immunoglobulin fusion proteins are similarto Ig but lack the variable regions and the CH1 domain, which have beenreplaced by the ligand-binding domain. Typically, the recombinantmolecule is generated at the cDNA level using recombinant DNA techniquesand expressed in cell culture. Most often the recombinant immunoglobulinfusion protein is a disulfide-linked homodimer. The variations on theabove described typical fusion protein are considerable. For example, inaddition to ligand-binding domains, many other fusion partners have beenplaced at the amino-terminus, such as ligands, enzymes, and peptideepitopes.

The polymers of the present invention contain an additional regioncomposed of linear polymers of at least portions of the hinge and CH2domain (HCH2 polymers), as described below. The length of the polymer isvaried. The introduction of a polymer unit between the framework domainand the amino-terminal domain in an IgG fusion protein results in amolecule that is tripartite in function. The framework domain providesfeatures common to IgG fusion proteins such as stability, covalentdimerization, single-step purification, and ease of detection (Chamow,et al., 1996). The intervening HCH2 polymer confers increased effectorfunction, including targeting to subsets of cells expressing FcR,increased capacity to ligate FcR, and to bind complement components. Theamino-terminal domain can deliver a second signal. Thus, multiplemolecular signals can be integrated into a single molecule with thepotential for synergistic interaction between the domains. The moleculesdescribed add a further dimension to IgG fusion protein platforms bypermitting the introduction of additional functions.

The polymers of this invention are composed of multiple HCH2 repeatunits. The polymers were developed using a cloning system that resultsin the rapid addition of HCH2 units into a human IgG₁ frameworkexpression vector. The HCH2 repeat unit is composed of the hinge and CH₂domain from an Ig such as IgG₁, which encompasses the region known tobind FcR and complement. To prevent inter-chain disulfide bond formationbetween the HCH2 units of the polymer, hinge region cysteines of theHCH2 monomer unit were mutated to serines. These mutations leave intactthose hinge residues known to interact with FcR and complement. Thehinge within the framework expression vector was not mutated thusretaining the dimeric structure of IgG. Several unique restriction siteson the 5′ end allow for the directional cloning of amino-terminaldomains into the polymer expression constructs.

In some embodiments of the invention, it is not necessary for theentirety of the HCH2 region to be employed in making the polymers. Asdescribed above, the entire human IgG1 HCH2 encompasses amino acidresidues 216 to 340 of the human IgG1 H chain (Eu numbering), with thehinge region spaning residues 216 to 237 and the CH2 domain encompassingresidues 238 to 340. The interactions between IgG and Fc receptors havebeen analyzed in biochemical and structural studies using wild type andmutated Fc. The consensus that has emerged from numerous studies is thatthe critical regions for binding to Fc receptors are located in the partof the hinge region closest to the CH2 domain and in the amino-terminusof the CH2 domain that is adjacent to the hinge. Of particularimportance are residues 233-239 (Glu-Leu-Leu-Gly-Gly-Pro-Ser). Mutationswithin this region result in substantially altered binding to Fcreceptors. This region is also responsible for many of the directinteractions with Fc receptors as determined by crystallographicstudies. Further into the CH2 domain, and away from the hinge, are otherresidues that may be, at least in some contexts, important for Fereceptor binding. Among them are Asn-297 and Pro-329. Pro-329 isinvolved in direct contact with Fc receptor. Asn-297 is the sole sitefor N-linked glycosylation within the Fc region. The presence ofcarbohydrate at this residue is crucial for binding to Fc receptors. Itmust be noted however that peptides spanning residues 233-239 of IgG1 Fcbind to FcγRIII poorly. Thus it may be argued that this region is mosteffective in engaging Fc receptor in the context of the overallstructure of the HCH2 region.

In the examples presented below the polymers were constructed using thehuman IgG1 HCH2 region which encompasses amino acid residues 216 to 340of the human IgG1 H chain. This region contains the sequences known tobe vital for Fc receptor binding as well as additional flankingresidues. The flanking residues provide structural stability and spacingbetween the HCH2 units. The inventors envisage that in some embodimentsit can be advantageous to construct HCH2 polymers comprised of regionswithin the HCH2 instead of the entire HCH2 unit. This may be done forexample to reduce the size of the HCH2 unit and hence the polymer. Oneway that this could be achieved is through the deletion of flankingresidues on either side of the region known to be vital for Fc receptorbinding. For instance the hinge could be truncated to span residues 233to 237 instead of residues 216 to 237 as used in the examples presentedherein. Similar considerations apply to the CH2 region which spansresidues 238-340 and to the hinge and CH2 regions of other Ig'sincluding IgA, IgD, IgG2, IgG3, IgG4, and IgE. Of course, those of skillin the art will, in view of the teachings of this specification, be ableto make, test, and use any number of different configurations ofportions of HCH2 regions in the context of the invention.

The polymers of this invention bind to low affinity FcR. In someinstances the polymers will bind the high affinity FcR receptors, forinstance the FcγRI receptor. This is a natural consequence of the highbinding affinity of the high affinity FcR receptors for the HCH2 region.

In some instances it can be advantageous to construct HCH2 polymers thatbind all forms of the low affinity FcγR receptors such as, for example,FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa and FcγRIIIb. In other embodimentsthe number and spacing of HCH2 units comprising the polymer are variedto increase the binding to one type of FcR receptor or conversely todecrease binding to another type of FcR receptor. In yet otherembodiments alterations to the HCH2 monomer unit can be made to increasespecificity of the polymer for one type of FcγR receptor and/or todecrease specific binding to another type of FcγR receptor. Suchalterations are achieved by mutating certain amino acid residues withinthe HCH2 sequence to other amino acid residues. The choice of residuesto mutate within the HCH2 unit is determined by choice of targetreceptor specificity and are well known to those skilled in the art. Forexample see Shields, et al., 2001; Sondermann, et al., 2000; Morgan, etal., 1995; Hulett, et al., 1994. In other embodiments the specificbinding of the HCH2 polymers to different FcγR receptors can be enhancedby the presence of—and type of glycosylation of the HCH2 polymer. Choiceof expression system in which to produce the HCH2 polymers in partdetermines the extent and type of glycosylation.

In the examples presented herein the polymers were constructed using DNAsequences from human IgG1. In some instances it can be advantageous toconstruct HCH2 polymers comprised solely of human sequences to use asimmunotherapeutic agents in humans. However in some embodiments thepolymers are assembled from sequences of other Ig's including IgA, IgD,IgG, IgM, and IgE. In other embodiments the polymers are assembled fromsequences of more than one type of Ig, for example a polymer containingHCH2 units derived from IgG sequences are linked to HCH2 units derivedfrom IgE sequences. In other embodiments the HCH2 polymers are comprisedof non-human sequences. The choice of sequences used to construct thepolymers and polymer fusion proteins is determined by the targetreceptor and host identity (human or non-human). In yet otherembodiments the hinge region cysteines are mutated to amino acidresidues other than serine. In some embodiments the HCH2 unit may bealtered and/or mutated to bind complement components and not to bind toFcR. In other embodiments the HCH2 unit may be altered and/or mutated tobind FcR and to not bind complement.

In the examples presented herein the polymers were constructed using DNAsequences from human IgG1. The expressed proteins have been evaluatedfor their interactions with low affinity FcγR receptors. However in someembodiments the polymers are assembled from sequences of other Ig'sincluding IgA, IgD, IgU, IgM, and IgE and these polymers will bind toand interact with the FcR for other Ig's including FcαR, Fcεt, FcμR,FcδR, and FcRn. In other embodiments the polymers are assembled fromsequences of more than one type of Ig, for example a polymer proteincontaining HCH2 units derived from IgG sequences and IgE sequences willinteract and bind with the FcR for more than one type of Ig.

In the examples presented herein the polymers are constructed frommonomers consisting of full length HCH2 units. In some embodiments itmay be advantageous to construct polymers that contain monomers that aresmaller than full length HCH2 units. HCH2 polymer proteins derived froma smaller HCH2 unit would have a smaller size and mass yet still retainthe ability to effectively bind to and activate FcR and/or complement.The reduction in the size of the HCH2 unit is achieved by the removal ofsequences that are not involved in the binding to FcR and/or complement.The identity of the sequences that are not involved in the binding toFcR and/or complement are well known, as are the methods for theirremoval from the HCH2 monomer unit. The removal of these sequences wouldfail to affect the desired binding but yield a polymer of smaller mass.

Recombinant HCH2 polymer constructs can mimic the biological activityand functions of immune complexes (ICs), of aggregated IgG (AIG), and ofaggregated Fc. The use of recombinant HCH2 polymer constructs offersseveral advantages over AIG or Fc aggregates. The number and spacing ofHCH2 units can be altered to hone interaction with FcR's. Aggregates areby nature heterogeneous with considerable variation between batcheswhereas the recombinant HCH2 polymers are precisely defined. As shownherein HCH2 polymers are considerably more potent than AIG. Perhaps thisresult is achieved by expressing only those determinants necessary forFcR engagement and/or by presenting them in a particularly favorableconfiguration.

The receptors can be specifically activated with constructs containingdifferent numbers of HCH2 units. As shown herein, the number ofrepeating HCH2 units available to bind receptor markedly influences cellfunction. Cell function changes with addition of a single HCH2 unit. Theconstructs of the present invention allow for the measurement of changein receptor function based on IC size. The number of repeating HCH2units included within the polymer construct is variable and is selectedin order to optimize biological activity. In one embodiment the HCH2polymers are assembled as disulfide-linked homodimers. In someembodiments the HCH2 polymers are assembled as monomers (single chainpolypeptide), or hetero- or homo-multimers, and particularly as dimers,tetramers, and pentamers.

The extracellular domain of a variety of proteins, including human CD8αand human serum albumin (HSA) can be expressed as HCH2 polymer fusionproteins. The biological activity of these recombinant CH2 polymerscompares favorably to the activity of AIG and an anti-CD16 monoclonalantibody. The activity of the fusion proteins positively correlates tothe number of HCH2 units. The largest polymer tested using either a CD8αextracellular domain or HSA domain was several times as potent as AIG atsimilar concentrations.

Many protein domains can be expressed as HCH2 polymer fusion proteins. Anonlimiting list of such proteins includes ligand-binding domains,extracellular domains of receptors, enzymes, adhesion molecules,cytokines, peptide hormones, immunoglobulin fragments (Fab′), ligands,and antigens. Sites at which the fusion of the protein domain are madeare well known and may be selected to optimize biological activity,stability, secretion, avidity, and binding specificity. HCH2 polymerfusion proteins involving IgG1 were designed using sequence data fromthe human IgG1 constant region gene as a guide (accession #Z17370). Twoamino terminal domains have been expressed fused to the HCH2 polymers:the extracellular domain of human CD8α (accession #M12824) and domain Iof human serum albumin (accession #V00494). In some instances it may beadvantageous to construct HCH2 polymers composed of the HCH2 polymerregion unfused to additional protein domains or framework sequences.

In certain preferred embodiments the fusion proteins of the currentinvention are produced by the insertion of the HCH2 polymeric regioninto an existing antibody sequence or the sequence of a recombinantprotein. This process is advantageous in that there is a large body ofresearch on numerous antibodies that can be used to bind to a preferredtherapeutic agent. Another advantage of this method is its simplicity.The HCH2 polymeric region is a discrete, modular DNA element designedfor easy transfer from one cDNA construct to another. A modular DNAelement is sometimes referred to as a ‘cloning cassette’. The HCH2polymeric region can be used as a cloning cassette and simply splicedinto the existing cDNA for any protein, thus removing several steps fromthe formation process. In certain circumstances the precise site ofinsertion within a protein sequence will be determined by routineexperimentation. However, the appropriate site of insertion forimmunoglobulins and proteins derived therefrom is well known. Using thisapproach, existing monoclonal antibodies and recombinant immunoglobulinfusion proteins can be easily modified through the addition of the HCH2polymer region.

C. THE IMMUNE SYSTEM

The immune system can be divided into two arms known as the innate andadaptive immune systems. The innate immune system provides a first lineof defense against invading microorganisms or other insults. Cell typesinvolved in innate system defenses include natural killer (NK) cells, Bcells responsible for natural antibody production, andmonocytes/macrophages. The adaptive immune system is more finely honed,exhibits immunological memory and provides a second and more specificline of defense. Cell types involved in adaptive immunity include Tcells, B cells involved in T cell-dependent antibody responses, andagain monocytes/macrophages. Interactions between the innate andadaptive systems are complex with reinforcement under some circumstancesand antagonism under others. Several of these interactions involvecontrols exerted by immune complexes and, by extension, by the presentinvention, inasmuch as the present invention provides a mimic for immunecomplexes.

1. NK Cells

NK cells are large granular lymphocytes that are generated in the bonemarrow. They can be recognized by their distinct morphology and bysurface marker molecules that they either express or fail to express. NKcells are CD16⁺, CD56⁺, and CD3⁻. NK cells are found in the spleen, theblood, and the tissues, but not in lymph nodes. They have the capacityto kill some tumor cells, some virally infected cells, and to releasecytokines, and in particular interferon gamma and tumor necrosis factor(TNF) both of which contribute to the initial control of viralinfections and both of which additionally exert regulatory influencesover the T and B cells involved in adaptive immunity and autoimmunediseases. The present invention activates NK cells causing them toproliferate and to secrete increased amounts of interferon gamma andTNF. The CD16 surface marker that NK cells express is the FcγRIIIareceptor to which the present invention binds. This binding isresponsible for the activation of NK cells. NK cells themselves have aprotective role in at least some autoimmune processes since theirdepletion augments the severity of experimental autoimmuneencephalomyelitis, an animal model for multiple sclerosis, one of themajor autoimmune diseases of man. Accordingly NK cell activation by thepresent invention offers the prospect of improved treatment forautoimmune processes.

It is one embodiment of the current invention that HCH2 polymer fusionproteins can be used to activate NK cells, drive them to expand innumber both in vivo and in vitro, and to secrete cytokines with knownanti-tumor and anti-viral activities for the purpose of targeting,eliminating, or otherwise destroying neoplastic cells, malignant cellsthat make up a tumor, cancer cells or virally infected cells. Culture ofhuman peripheral blood lymphocytes (PBL) with high doses of IL-2 resultsin the generation of cytotoxic cells termed lymphokine-activated killer(LAK) cells (Rosenstein, M., et al., 1984). LAK cells are a mixture ofNK cells and T cells. A characteristic of LAK cells is their ability tolyse a variety of tumor cells in a non-MHC-restricted fashion. Tumorcell recognition by NK cells is antigen independent. One approach tocancer therapy is to administer high doses of intravenous IL-2 togenerate NK/LAK cells from PBL in vivo. However the substantial toxicityof therapeutic levels of intravenous IL-2 have limited its use as acancer therapeutic. Consequently NK/LAK cells are cultured from patientPBL in vitro with high doses of IL-2 and, after several cycles ofexpansion in cell number, returned to the patient (Hayes, R., et al.,2001). A related approach involves the isolation of tumor infiltratinglymphocytes (TIL) from resected tumors which are also expanded in vitrowith high doses of IL-2. Though they represent a small fraction of thetotal immunocyte population, activated NK cells are an importantcomponent of TIL (Brittenden, J., et al., 1996). The polypeptides of thecurrent invention, in conjunction with IL-2, are potent activators of NKcells. NK cells are activated by HCH2 polymers to a much higher levelthan is possible with IL-2 alone (Example 13). HCH2 polymers offer twodistinct advantages over IL-2 therapy alone. First, co-administration ofHCH2 polymers and IL-2 may achieve NK cell activation at substantiallylower levels of IL-2 than when IL-2 alone is employed thus avoiding manyof the toxicities associated with high level IL-2 therapy in vivo. TheHCH2 polymers of the current invention are anticipated to findapplication for the in vitro expansion of LAK/TIL cells as well.Secondly, the HCH2 polymers also potently induce the secretion of TNF-αand IFN-γ from NK cells (Example 16). Both TNF-α and IFN-γ have knownanti-tumor and anti-viral effects (Fortis, C., et al., 1999).

2. Monocytes and Macrophages

Macrophages derive from monocytes and share many of their functions. Forthis reason the two will be considered together. Monocytes mature in thebone marrow. They are found in the blood and throughout the tissues.They function both in innate defenses and in adaptive defenses. At theonset of viral infections they release IL-12, a protein that activatesNK cells for interferon gamma and TNF production, the importance ofwhich in viral and tumor defenses has been discussed above. Monocytesfunction as antigen presenting cells and for this reason are criticalfor the activation of T cells and hence adaptive immunity. They, inturn, are activated for phagocytosis by products released by activated Tcells and, once activated, clear both invading organisms, and cells andtissues damaged by invading organisms or by autoimmune processes.

Monocytes also exhibit regulatory properties exerted by products thatthey release. These products act to inhibit immune responses includingautoimmune responses. Among the regulatory molecules released bymonocytes are IL-10 and prostaglandin E2.

Monocytes express all three classes of Fc receptor. Binding of HCH2polymers to FcR expressed on monocytes may result in the ligation of FeγRII, the ligation of FcyγIIIa or in the coaggregation of both FcγRIIIand FcγRIIIa receptors. Ligation of the FeγRIIIa receptor on monocytesis known to potently induce IL-10 and prostaglandin E2 production bymonocytes (Passwell, et al., 1979; Ferreri, et al., 1986; Berger, etal., 1996) offering the prospect that the present invention may, viasimilar induction, contribute to the control of autoimmune processes.

3. B Cells

B cells produce antibody. Initial production of IgM class antibody isindependent of T cell influence but the subsequent switch to IgG andother classes of antibody occurs under the direction of T cells. Both Tcell-independent antibodies and T cell-dependent antibodies canparticipate in autoimmune responses. B cells express the FcγRIIbreceptor (CD32). The FcγRIIb receptor delivers a negative signalmediated through a specialized signaling motif know as theimmunoreceptor tyrosine-based inhibitor motif (ITIM) located within thecytoplasmic tail of the receptor. The ITIM motif is a unique feature ofthe FcγRIIb receptor and is not present in any other Fey receptor class.Ligation of this receptor provides a negative signal to B cells andhence an inhibitory signal for antibody production. The FcγRIIb receptorrecognizes and responds to IgG-containing immune complexes and to IgGaggregates. Accordingly, it would be expected to respond to the presentinvention with, as a consequence, inhibition of production of both IgMclass and IgG class immunoglobulin production.

Antibody production by B cells is also subject to down-regulatorycontrols exerted by products released by T cells and monocytes. Amongsuch controls is that exerted by regulatory CD8 cells, sometimesreferred to as suppressor cells. IgG aggregates, added to peripheralblood mononuclear cell preparations in vitro, activate CD8 cell-mediatedinhibition of immunoglobulin production, even though CD8 cells do notexpress the Fcγ receptors to which IgG aggregates bind. The result isthought to depend on an induction of CD8 cell activity by productsreleased by the NK cells and monocytes to which IgG aggregates do bind.Since the present invention mimics the activity of IgG aggregates, anindirectly mediated induction of CD8 cell-mediated regulatory activity,and hence a beneficial effect on autoimmunity was anticipated and infact found.

4. T Cells

T cells are small lymphocytes that mature in the thymus, whence theymake their way to the lymphoid organs. T cells can be divided into 2major categories known as CD4 cells and CD8 cells. The two categorieshave different, albeit overlapping, functions. CD4 cells are responsiblefor cell-mediated immunity, sometimes referred to as delayed typehypersensitivity. Cell-mediated immunity is implicated in thepathogenesis of numerous autoimmune diseases. CD4 cells recognizeantigens by means of T cell receptors expressed on the cell surface.Antigenic peptide fragments are presented to CD4 cells by MHC class IImolecules expressed on the surface of antigen presenting cells such asmonocytes. T cells are activated by this presentation but only if asecond activating signal is provided by the antigen presenting cell.Activated CD4 cells release cytokines that are responsible for theirbiologic effects. CD4 cells can be divided into 2 subclasses known asTh1 cells and Th2 cells. Th1 cells are responsible for cell-mediatedimmunity while Th2 cells direct production of IgG and other classes ofimmunoglobulin by B cells.

CD4 cell responses are tightly regulated and the regulatory mechanismscan be exploited to treat autoimmune diseases. CD4 cells do not expressFcγ receptors but a control over CD4 cells can be exerted by cytokinessuch as IL10 produced by monocytes as a consequence of Fcγ receptorligation. Cytokines released by NK cells as a sequelum of Fcγ receptorligation can also contribute to CD4 cell inactivation. In addition,products released by CD8 cells (see below) can inhibit CD4 cells. CD8cells are primed for regulatory activity by products released by NKcells and monocytes following ligation of the Fcγ receptors expressed onthe surface of NK cells and monocytes. For all these reasons the presentinvention offers the prospect of inhibiting those activities of Th1 andTh2 cells that contribute to the pathogenesis of autoimmune processes.

CD8 cells comprise the second major category of T cells. They recognizepeptides presented to them by MHC class I molecules expressed on thesurface of antigen presenting cells. As with CD4 cells a co-stimulatorysignal is required for CD8 cell activation. The vast majority of CD8cells do not express Fcγ receptors. CD8 cells have 2 established majorfunctions. The first is cytotoxicity, a critical component in thecontrol of infections by viruses and other organisms that resideintracellularly. The second major function is the regulatory functionalready discussed.

The brief synopsis of immune system function given above is cursory. Itspurpose is to highlight some of the mechanisms by which the presentinvention can be expected to have a favorable impact on autoimmuneprocesses.

D. Fc RECEPTORS AND THE COMPLEMENT SYSTEM

There are three classes of Fc receptor (Gessner et al., 1998; Raghavanet al., 1996). FcγRI (CD64) binds monomeric IgG with high affinitywhereas AIG and IC bind preferentially to FcγRII (CD32) and FcγRIII (CD16), the low affinity receptors for Fc. FcγRII and FcγRIII are closelyrelated in the structure of their ligand-binding domains. In humansthree separate genes, FcγRIIA, FcγRIIB, and FcγRIIC, two of which giverise to alternatively spliced variants, code for FcγRII. FcγRIIadelivers activating signals whereas FcγRIIb delivers inhibitory signals.The functional basis for the divergent signals arises from signalingmotifs located within the cytoplasmic tails of the receptors. Animmunoreceptor tyrosine-based inhibitor motif (ITIM) located in thecytoplasmic tail of the FcγRIIb is critical for negative receptorsignaling. The ITIM motif is a unique feature of the FcγRIIb receptorand is not present in any other Fcγ receptor class. In contrast, anactivatory immunoreceptor tyrosine-based activation motif or ITAM islocated in the cytoplasmic tail of FcγRIIa. ITAM motifs transduceactivating signals They are also found in the FcR γ-chains, which areidentical to the γ-chains of the high affinity IgE receptor (FcεRI).While FcγRIIa and FcγRIIb are widely expressed on myeloid cells and someT-cell subsets they are notably absent from NK cells.

Human FcγRIII is also present in multiple isoforms derived from twodistinct genes (FcγRIIIA and FcγRIIIB). FcγRIIIb is unique in itsattachment to the cell membrane via a glycosylphosphatidyl anchor.FcγRIIIb expression is restricted to neutrophils while FcγRIIIa isexpressed by macrophages, and NK cells (both FcγRIIIa). FcγRIIIa is alsoexpressed by some γδ T-cell subsets and certain monocytes. FcγRIIIarequires the presence of the FcR γ-chain and/or the CD3 ζ-chain for cellsurface expression and signal transduction. The FcR γ-chain and the CD3ζ-chain are dimeric and possess ITAM motifs. FcγRIIIa forms a multimericcomplex with these subunits and signaling is transduced through them.Thus, there is considerable FcγR receptor heterogeneity and diverseexpression profiles.

AIG and IC have been used to target FcRIIIa on immune cells, but asnoted earlier production of defined AIG and IC was seen to beproblematic. Assembly of complexes by physical or chemical methods isdifficult to control with precision resulting in heterogeneity withincomplexes of similar molecular weight in addition to variations betweenpreparations and changes in composition upon storage. Molecular cloninghas been used in the present invention to create molecules that canmimic or approximate AIG and IC function with respect to theirinteractions with FcR and which allow for the inclusion and targeting ofa second protein domain to cells expressing FcR.

The binding sites for FcγRII and FcγRIII map to the hinge and proximalregion of the CH2 domain of IgG, the same region originally identifiedfor FcγRI (Duncan et al., 1988; Morgan et al., 1995; Lund et al., 1991).White et al. (2001) describe the cloning and expression of linearpolymers of the hinge and CH2 (HCH2) fused to the Fc region of IgG₁ anddemonstrate their biological activity. Legge et al. (2000) have recentlyshown that an aggregated PLP1 immunoadhesin, unlike the monomeric form,moderates disease severity in experimental autoimmune encephalomyelitis,the rodent model for multiple sclerosis. This change is due to the dualfunctionality of the aggregated Fc and PLP moieties within the complex.

In the later phase of a primary immune response or in chronic responses,large ICS form. These complexes signal through the low affinity IgGreceptors that recognize ICS or IgG aggregates preferentially. The lowaffinity receptors are of two classes FcγRII (CD32) and FcγRIII (CD15).FcγRIIb provides an inhibitory signal for secretion of cytokines thataugment immunoglobulin secretion including IgG secretion. FcγRIIIa(found on NK cells, monocytes and γδ T cells) preferentially recognizesIgG1. One thrust of this invention is directed towards activation ofFcγRIIIa.

The ability of FcγR to bind IgG and transmit a signal into the celldepends upon the FcγRs alleles expressed, upon glycosylation, and howthe receptor is associated with the signaling subunit. In addition,glycosylation patterns differ between cell types and this too can affectligand binding to FcγRIIIa. FcγRIIIa on NK cells is glycosylated withhigh mannose oligosaccharides, whereas monocyte/macrophage FcγRIIIa isnot. Perhaps this imparts lower receptor affinity to monocyte/macrophageFcγRIIIa relative to NK cell FcγRIIIa, adding yet another level ofmodification to receptor function (Galon et al., 1997; Edberg et al.,1997). Thus, FcγR function is regulated at several levels, which canhave an impact on ligand binding and receptor signaling.

Recently, the inventors have initiated studies into the potentialimmunomodulatory role of immune complexes (IC) in human autoimmunesyndromes. Central to these studies are the interactions between IC andFcR. However, production of defined IC is difficult to control withprecision. Molecular cloning is used to create molecules that can mimicor approximate IC function with respect to their interactions with FcRand which allow for the inclusion and targeting of a second proteindomain to cells expressing FcR. The strategy pursued is to expressmultiple linear copies of the region of the IgG framework that bindsFcR. Expressing only those determinants necessary for FcR engagement andpresenting them in a particularly favorable configuration results innovel proteins that are considerably more potent than IC. Thusrecombinant IC mimetic proteins described herein will provide both avaluable tool for the examination of IC deposition and in thetherapeutic targeting of FcR in autoimmune disorders.

1. FcγRIIIa and NK Cells

NK cells express only one type of FcγR, FcγRIIIa, which upon ligationresults in lymphokine expression and up-regulation of the constitutivelyexpressed low affinity IL-2 receptor, IL-2R (p75) (Anegon, et at, 1988).Exposure to high dose IL-2 alone results in lymphokine expression,increased IL-2R expression, enhanced cytotoxic function, but only modestproliferative responses by NK cells (Nagler, et al., 1990).Co-stimulation with high doses of IL-2 together with FcγRIIIa ligationresults in greater NK-cell activation including brisker proliferationthan is achieved with IL-2 alone (Harris, et al., 1989). Theproliferative response of NK cells is used as a biological read-out forFcγR-ligand interactions. For example four HCH2 polymer constructs weretested on PBMC at 2.5 μg/mL and results were compared to those obtainedwith anti-CD16 mAb and AIG. As expected anti-CD16 mAb and AIG induced aproliferative response in the presence of IL-2. Proliferative responsedriven by the HCH2 polymer constructs correlated with the number ofrepeat units, CD8R0 gave a 7.5 fold induction over IL-2 alone whereasCD8R4 gave a 15-fold induction. CD8R4 induced the highest level ofactivation, but the level of induction by CD8R3 approached that forCD8R4 and the difference between them was not statistically significant.Both constructs may be at or near the number of HCH2 units necessary tooptimally engage FcγR. Direct comparison between CD8R4 and AIG revealsthat CD8R4 is considerably more potent that AIG: CD8R4 produces a18-fold induction at 5 μg/μL while 125 μg/μL of AIG is necessary toproduce similar levels of induction.

The availability of a recombinant protein that approximates thefunctions of aggregated IgG and at the same time delivers a secondsignal may permit dissection of the relative importance of the variousmechanisms that have been postulated to explain the immunomodulatoryrole of AIG. Blockade of FcγR probably does not suffice to explain themultiple effects of AIG on immune cells. Other potential mechanismsinvolve the inhibition of antigen recognition by T-cells, activation ofFcγRIII on NK-cells, and altered cytokine synthesis and secretion. HCH2polymers may also provide a valuable tool for the examination of ICdeposition.

Binding of ligands to FcγRIIIa on NK cells induces IFN-γ and TNFαsecretion, antibody dependent cellular cytotoxicity (ADCC),proliferation, and under some circumstances, but not all, apoptosis.Some functions require a second signal, which can be provided by IL-2.FcγRIIIa on NK cells can associate with either the γ chain of the highaffinity IgE receptor (FcεRI) or with the ζ chain of the T cell receptor(Lanier, et al., 1989; Lanier, et al., 1991; Kurosaki, et al., 1991).Either chain may transmit intracellular signals following ligand bindingto FcγRIIIa.

Signaling through the CD2 receptor on NK cells also occurs through the ζchain (Vivier, et al., 1991; Moingeon, et al, 1992) and anti-CD2antibodies have been effectively employed to study NK cell function andsuppressor mechanisms. Thus, activation of NK cells through CD2 or CD16induces overlapping effector functions, including secretion of IFN-γ andTNFα, and proliferation. Activation of NK cells through CD2 inducessecretion of TGFβ, which acts upon CD8 T cells that then inhibit Igsecretion by B cells (Gray, et al., 1994; Ohtsuka, et at, 1998). Releaseof soluble CD16, a powerful inhibitor of Ig secretion in vitro, byactivated NK cells provides a potential second mechanism of Igregulation by NK cell activation.

2. The Complement System

Signaling through the CD2 receptor on NK cells also occurs through the ζchain (Vivier, et al., 1991; Moingeon, et al, 1992) and anti-CD2antibodies have been effectively employed to study NK cell function andsuppressor mechanisms. Thus, activation of NK cells through CD2 or CD16induces overlapping effector functions, including secretion of IFN-γ andTNFα, and proliferation. Activation of NK cells through CD2 inducessecretion of TGFβ, which acts upon CD8 T cells that then inhibit Igsecretion by B cells (Gray, et al., 1994; Ohtsuka, et al., 1998).Release of soluble CD16, a powerful inhibitor of Ig secretion in vitro,by activated NK cells provides a potential second mechanism of Igregulation by NK cell activation.

The classical activation pathway and CDCC are the complement-mediatedmechanisms most relevant to the fusion protein of the current invention.The complement binding site on the Fc portion of IgG1 encompasses thehinge and CH2 regions. The inventors envisage the use of the HCH2polymers of the current invention to greatly facilitate classicalpathway and CDCC mediated removal of target cells. In othercircumstances complement activation may pose unwanted and problematicside reactions. In these cases the inventors envisage the alteration ofHCH2 polymer-C1q interactions so as to lessen or eliminate complementactivation.

E. USE OF HCH2 POLYMERIC FUSION PROTEINS AS IN VIVO AND IN VITROIMMUNOLOGICAL AGENTS

1. Clinical Use of Antibodies

The advent of monoclonal antibody (mAb) technology provided the basisfor developing potentially therapeutic reagents that react with specificcell surface antigens involved in cell function. Therapeutic reagents ofthis type can be incorporated into the fusion proteins of the currentinvention.

One of the clinically successful uses of monoclonal antibodies is tosuppress the immune system, thus enhancing the efficacy of organ ortissue transplantation. U.S. Pat. No. 4,658,019 describes a novelhybridoma (designated OKT3), which is capable of producing a monoclonalantibody against an antigen found on essentially all normal humanperipheral T cells. This antibody is said to be monospecific for asingle determinant on these T cells, and does not react with othernormal peripheral blood lymphoid cells. The OKT3 mAb described in thatpatent is currently employed to prevent renal transplant rejection(Goldstein, 1987).

Monoclonal antibodies are an emerging class of powerful therapeuticagents. Several have been approved for the treatment of malignanciesincluding cancer and many more are in the process of clinicaldevelopment. For example Rituximab (Rituxan, Mabthera) is a mAb whichtargets the CD20 molecule expressed on the surface of B cells. Rituximabwas the first therapeutic mAb approved for the treatment of amalignancy, in this case non-Hodgkin's lymphoma. Rituximab is a chimericIgG1 mAb composed of murine variable domains and human constant regions.Rituximab exerts its effects through several mechanisms: Induction ofapoptosis in B cells, direct complement killing (CDC) and cellulareffector mechanisms such as antibody-dependent cell-mediatedcytotoxicity (ADCC) and the related pathway of complement-dependentcellular cytotoxicity (CDCC) (Johnson, et al., 2001). CDC and cellulareffector mechanisms are mediated through the Fc region of mAb, inaddition ADCC relies on Fe-FcR receptor interactions. Two lines ofevidence strongly implicate interactions between Rituximab and theFcγRIIIa receptor as crucial to the therapeutic effectiveness ofRituximab. First, patients with the FcRγIIIa receptor 158V allotype havesubtantially greater clinical responses to Rituximab therapy, includingcomplete clearance of malignant cells, than patients with the FcγRIIIareceptor 158F allotype. The FcγRIIIa receptor 158V allotype has higheraffinity for human IgG1 than the FcγRIIIa receptor 158F allotype. Italso promotes increased ADCC (Cartron, et al., 2002). Secondly, micewith gene knock-outs that result in the loss of FcγRIIIa and FcγRIreceptor expression have deficient responses to anti-CD20 mAb therapies,including Rituximab (Clynes, et al, 2000).

a. Modification of Recombinant Monoclonal Antibodies by the Introductionof a HCH2 Polymer

It is an embodiment of the current invention that recombinant monoclonalantibodies (mAb) can be modified by the introduction of one or more HCH2units into the Fc region to create a HCH2 polymer of appropriate lengthwithin a monoclonal antibody. Monoclonal antibodies modified in thismanner will retain their target specificity while acquiring improvedand/or more selective effector function. HCH2 polymers greatly enhanceFe-FcR receptor interactions. More specifically HCH2 polymers of thecurrent invention have greatly improved binding to and enhancedactivation of FcγRIIIa receptors over that seen with the Fc portion ofmAb in current therapeutic use. As enhanced interaction of mAB withFcγRIIIa has been documented to have therapeutic benefit in thetreatment of malignancies the inventors envisage modifying existing mAbwith the introduction of an HCH2 polymer into the Fc region of the mAb.Monoclonal antibodies with this modification will have enhancedinteraction with FcγRIIIa receptors.

Functional IgG genes, those that direct expression of a mAb, arecomposed of heavy and light chain genes segments. Light chain (L) genesconsist of three exons, containing the hydrophobic leader sequence, thevariable regions and the L constant region (C_(L)). Separating the exonsare the intervening sequences or introns. Similarly, the variable regionof a functional Ig heavy chain (H) gene has a separate exon for each ofthe leader sequence, the variable region, and H chain constant region(CH1). The H gene also contains the Fc region which is composed ofseparate exons for the hinge, the CH2 region and CH3 regions. Once againthe exons are separated by introns. The expression of mAb in mammaliancells typically involves cloning both the H and L gene segments fromfunctional Ig genes into either a single expression vector or separateexpression vectors (one for L, one for H genes) that posses the Igpromoter region. Once subcloned the expression vectors possessing the Land H genes are transfected into an appropriate cell line forexpression. The use of gene segments insures the presence of intronicsequences, which contain enhancer and other elements that collectivelyallow for high levels of Ig expression in B cells and myeloma cells. Igexpression systems utilizing the Ig promoter and intronic geneticelements limit protein expression to cells of lymphoid derivationhowever.

More recently, Ig expression systems have been developed that use viralpromoters and enhancer combinations, such as CMV. The use of viralpromoter/enhancer combinations permits strong expression in bothlymphoid and non-lymphoid cells lines such as CHO and COS (Norderhaug,et al., 1997). Inclusion of the intronic enhancer from the Ig H genealso directs high level expression in lymphoid cells. Additionally, Hand L gene segments are no longer necessary for efficient expression andcan be replaced by their corresponding cDNA's (McLean, et al., 2000).

The introduction of HCH2 polymers into mAb can be achieved by any ofseveral approaches. In one method, using molecular cloning techniqueswell known to those skilled in the art, H chain gene segments withinexpression vectors are modified by the insertion of the HCH2 polymercloning cassette into the 5′ end of the hinge exon. The modified hingeexon now consists of the HCH2 polymer fused in frame to the hingesequences. The vector containing the modified H gene is introduced inconjunction with a L gene into an appropriate cell line for mAbexpression. A more preferable method is to replace the Fc gene segmentwith a cDNA segment comprising a splice acceptor signal, the HCH2polymer fused to an Ig Fc cDNA and a polyA signal. The modified H geneis then transferred into an Ig expression vector capable of directing Igexpression without Ig gene intronic sequences. The vector containing themodified H gene is introduced in conjunction with an L gene into anappropriate cell line for expression.

The insertion of HCH2 polymers into mAb expressed from cloned cDNAwithin expression vectors can also be achieved using similar techniques.For instance, the cDNA encoding the Fc region can be removed from the Hchain cDNA and replaced with a DNA segment encoding the HCH2 polymerfused to a Fc cDNA. Conversely, the cDNA encoding the H chain leader,variable and CH1 region can be excised and transferred to vectorscontaining the HCH2 polymer region genetically fused to a Fc cDNA.Alternatively, the HCH2 polymer cassette can be introduced into the Hchain cDNA at the appropriate site. This site would most commonly be thejunction between the CH1 region and the hinge. The method ofintroduction is well known to those skilled in the art. Subsequently,the modified H chain cDNA is then transferred into an Ig expressionvector capable of directing Ig expression without Ig gene intronicsequences. The vector containing the modified H chain cDNA is introducedin conjunction with an L chain expression vector into an appropriatecell line for expression.

While interaction with FcγRIIIa receptors is important for the efficacyof several mAb in clinical use, the methods of modification describedabove are general. In other applications, HCH2 polymers can beintroduced into mAb to enhance specificity for other individual FcRreceptors, classes of FcR receptors, as blocking reagents for FcRreceptors, or for binding to complement factors.

2. Preparation of Monoclonal and Polyclonal Antibodies

The methods for generating monoclonal antibodies (MAbs) generally beginalong the same lines as those for preparing polyclonal antibodies.Briefly, one begins by immunizing an animal with an immunogen, andcollecting antisera from that immunized animal to prepare a polyclonalantibody. A wide range of animal species can be used for the productionof antisera. Typically an animal used for production of antisera is arabbit, a mouse, a rat, a hamster or a guinea pig. Because of therelatively large blood volume of rabbits, a rabbit is a preferred choicefor production of polyclonal antibodies.

As is well known in the art, a given polypeptide or polynucleotide mayvary in its immunogenicity. It is often necessary therefore to couplethe immunogen with a carrier. Exemplary and preferred carriers arekeyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Otheralbumins such as ovalbumin, mouse serum albumin or rabbit serum albumincan also be used as carriers.

Means for conjugating a polypeptide or a polynucleotide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As is also well known in the art, immunogenicity to a particularimmunogen can be enhanced by the use of non-specific stimulators of theimmune response known as adjuvants. Exemplary and preferred adjuvantsinclude complete Freund's adjuvant, incomplete Freund's adjuvant andaluminum hydroxide adjuvant.

The amount of immunogen used for the production of polyclonal antibodiesvaries inter alia, upon the nature of the immunogen as well as theanimal used for immunization. A variety of routes can be used toadminister the immunogen (subcutaneous, intramuscular, intradermal,intravenous and intraperitoneal). Blood of the immunized animal issampled at various time points following immunization to monitor theproduction of polyclonal antibodies. When a desired level ofimmunogenicity has been obtained, the immunized animal can be bled andthe serum isolated and stored.

A monoclonal antibody can be readily prepared through use of well-knowntechniques such as those exemplified in U.S. Pat. No 4,196,265, hereinincorporated by reference. Typically, a technique involves firstimmunizing a suitable animal with a selected antigen (e.g., apolypeptide or polynucleotide of the present invention) in a mannersufficient to provide an immune response. Rodents such as mice and ratsare preferred animals. Spleen cells from the immunized animal are thenfused with cells of an immortal myeloma cell. Where the immunized animalis a mouse, a preferred myeloma cell is a murine NS-1 myeloma cell.

The fused spleen/myeloma cells are cultured in a selective medium toselect fused spleen/myeloma cells from the parental cells. Fused cellsare separated from the mixture of non-fused parental cells, for example,by the addition of agents that block the de novo synthesis ofnucleotides in the tissue culture media. Exemplary and preferred agentsare aminopterin, methotrexate, and azaserine. Aminopterin andmethotrexate block de novo synthesis of both purines and pyrimidines,whereas azaserine blocks only purine synthesis. Where aminopterin ormethotrexate is used, the medium is supplemented with hypoxanthine andthymidine as a source of nucleotides. Where azaserine is used, themedium is supplemented with hypoxanthine.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants for reactivitywith antigen-polypeptides. The selected clones can then be propagatedindefinitely to provide the monoclonal antibody.

By way of specific example, to produce a monoclonal antibody, mice areinjected intraperitoneally with between about 1-200 μg of an antigencomprising a polypeptide. B lymphocytes are stimulated to grow byinjecting the antigen in association with an adjuvant such as completeFreund's adjuvant (a non-specific stimulator of the immune responsecontaining killed Mycobacterium tuberculosis). At some time (e.g., atleast two weeks) after the first injection, mice are boosted byinjection with a second dose of the antigen mixed with incompleteFreund's adjuvant.

A few weeks after the second injection, mice are tail bled and the seratitered by immunoprecipitation against radiolabeled antigen. Preferably,the process of boosting and titering is repeated until a suitable titeris achieved. The spleen of the mouse with the highest titer is removedand the spleen lymphocytes are obtained by homogenizing the spleen witha syringe. Typically, a spleen from an immunized mouse containsapproximately 5×10⁷ to 2×10⁸ lymphocytes.

Mutant lymphocyte cells known as myeloma cells are obtained fromlaboratory animals in which such cells have been induced to grow by avariety of well-known methods. Myeloma cells lack the salvage pathway ofnucleotide biosynthesis. Because myeloma cells are tumor cells, they canbe propagated indefinitely in tissue culture, and are thus denominatedimmortal. Numerous cultured cell lines of myeloma cells from mice andrats, such as murine NS-1 myeloma cells, have been established.

Myeloma cells are combined under conditions appropriate to foster fusionwith the normal antibody-producing cells from the spleen of the mouse orrat injected with the antigen/polypeptide. Fusion conditions include,for example, the presence of polyethylene glycol. The resulting fusedcells are hybridoma cells. Like myeloma cells, hybridoma cells growindefinitely in culture.

Hybridoma cells are separated from unfused myeloma cells by culturing ina selection medium such as HAT medium (hypoxanthine, aminopterin,thymidine). Unfused myeloma cells lack the enzymes necessary tosynthesize nucleotides from the salvage pathway because they are killedin the presence of aminopterin, methotrexate, or azaserine. Unfusedlymphocytes also do not continue to grow in tissue culture. Thus, onlycells that have successfully fused (hybridoma cells) can grow in theselection medium.

Each of the surviving hybridoma cells produces a single antibody. Thesecells are then screened for the production of the specific antibodyimmunoreactive with an antigen/polypeptide. Limiting dilution of thehybridomas isolates single cell hybridomas. The hybridomas are seriallydiluted many times and, after the dilutions are allowed to grow, thesupernatant is tested for the presence of the monoclonal antibody. Theclones producing that antibody are then cultured in large amounts toproduce an antibody in convenient quantity.

3. In Vitro Uses of HCH2 Polymeric Fusion Proteins

In addition to the above-described uses, the claimed peptides will havea variety of in vitro uses. Some of these are described below; those ofskill in the art will understand others.

a. Immunoassays

The success of immunoassays that accurately quantitate the amount of adesired agent is based in large part on either capturing the agent in aspecific manner, detecting the agent in a specific manner, or both. Theinventors envisage that the fusion protein of the current invention mayfind utility in the specific detection of, or capturing of, Fcreceptors. The binding of the fusion proteins of the current inventionto Fc receptors allows for their use in the specific capturing ordetecting of these receptors. The fusion proteins of the currentinvention may be immobilized onto a suitable surface and used to captureFc receptors. Subsequently, the captured Fc receptors may be detectedusing other agents such as antibodies directed against a noncompetingsite of the receptor. Alternatively, the fusion proteins may be used todetect Fc receptors that are either nonspecifically or specificallycaptured onto a suitable surface. By varying the immunoassay procedurethe fusion proteins of the current invention may be utilized for thedetection of CD16 and CD32 in a variety of manners.

The inventors further envisage that the fusion proteins of the currentinvention will find utility in the detecting or capturing of a widerange of agents. Immunoassays allow for the detection and quantitationof an agent, and in particular the presence and quantitation of a smallamount of agent, by including a step in the procedure that amplifies thesignal to noise ratio. The fusion proteins of the current invention canbe used to amplify the signal to noise ratio in a wide variety ofimmunoassays by virtue of their repetitive HCH2 regions. In thisembodiment of the current invention, a fusion protein would coexpress aligand binding domain able to bind specifically to the agent of interestgiving specificity to the fusion protein. The repetitive HCH2 regionallows for an amplification step as it contains numerous repeating unitsthat can be targeted by a wide variety of agents known to those familiarin the art. For example, polyclonal sera, conjugated to an enzyme orother suitable signal generating agent, reactive with the HCH2 regionmay be used in the detecting procedure. The repeating HCH2 region wouldallow for the binding of numerous Ig present in the polyclonal sera thatare reactive with the HCH2 region. The fusion proteins of the currentinvention may find utility in the detection of agents in a wide varietyof immunoassays by varying the ligand binding proteins coexpressed inthe fusion proteins. A non-limiting list of agents that may be detectedwith the aid of the fusion proteins of the current invention include,cytokines, soluble receptors, steroids, soluble proteins, and hormones.Variations of immunoassay procedure are envisaged by the inventors andshould be known to those familiar in the art. Although the abovementioned uses of the fusion proteins of the current invention arediscussed in the context of immunoassays, the authors envisage that theyare readily applicable to numerous in vitro uses where the detection ofa specific agent is desired. A nonlimiting list of such in vitro usesinclude their use in fluorescence activated cell sorting,immunohistochemistry, and immunoprecipitation.

The fusion proteins of the invention will find utility in immunoassaysfor the detection of CD16. Turning first to immunoassays, in their mostsimple and direct sense, preferred immunoassays of the invention includethe various types of enzyme linked immunosorbent assays (ELISAs) knownto the art. However, it will be readily appreciated that the utility ofantibodies is not limited to such assays, and that other usefulembodiments include RIAs and other non-enzyme linked antibody bindingassays or procedures.

In the preferred ELISA assay, samples to be tested for CD16 areimmobilized onto a selected surface, preferably a surface exhibiting aprotein affinity such as the wells of a polystyrene microtiter plate.After washing to remove incompletely adsorbed material, one will desireto bind or coat onto the well a nonspecific protein such as bovine serumalbumin (BSA), casein or solutions of milk powder that is known to beantigenically neutral with regard to the fusion protein. This allows forblocking of nonspecific adsorption sites on the immobilizing surface andthus reduces the background caused by nonspecific binding of theantibody onto the surface.

After binding of antigenic material to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with a fusionprotein of the current invention in a manner conducive to immune complex(antigen/antibody) formation. Such conditions preferably includediluting with diluents such as BSA, bovine gamma globulin (BGG) andphosphate buffered saline (PBS)/Tween. These added agents also tend toassist in the reduction of nonspecific background. The layered antibodyis then allowed to incubate for from 2 to 4 hours, at temperaturespreferably on the order of 22° to 25° C. Following incubation, theantibody-contacted surface is washed so as to remove non-immunocomplexedmaterial. A preferred washing procedure includes washing with a solutionsuch as PBS/Tween, or borate buffer.

Following formation of specific immunocomplexes between the fusionprotein and the bound antigen, and subsequent washing, the occurrenceand amount of immunocomplex formation may be determined by subjectingsame to a second antibody having specificity for the fusion protein ofthe current invention. Of course, in that the fusion protein willtypically have a human IgG region, the second antibody will preferablybe an antibody having specificity in general for human IgG. To provide adetecting means, the second antibody will preferably have an associatedenzyme that will generate a color development upon incubating with anappropriate chromogenic substrate. Thus, for example, one will desire tocontact and incubate the antisera-bound surface with a urease orperoxidase-conjugated anti-human IgG for a period of time and underconditions which favor the development of immunocomplex formation (e.g.,incubation for 2 hours at room temperature in a PBS-containing solutionsuch as PBS-Tween).

After incubation with the second enzyme-tagged antibody, and subsequentto washing to remove unbound material, the amount of label is quantifiedby incubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS]and H₂O₂, in the case of peroxidase as the enzyme label. Quantificationis then achieved by measuring the degree of color generation, e.g.,using a visible spectrum spectrophotometer.

b. Fluorescence Activated Cell Sorting (FACS)

Fluorescent activated cell sorting; flow cytometry or flowmicrofluorometry provides a means for scanning of individual cells forthe presence of an antigen. The method employs instrumentation that iscapable of activating, and detecting, the excitation emissions oflabeled cells in a liquid medium.

FACS is unique in its ability to provide a rapid, reliable,quantitative, and multiparameter analysis of either living or fixedcells. The peptides of the current invention provide a useful tool forthe analysis and quantitation of antigenic, biophysical, and biochemicalcharacteristics of individual cells. When used with electrostaticdeflection technology, the fusion proteins of the present invention canbe used for the specific isolation of subpopulations of cells.

c. Immunohistochemistry

The fusion proteins of the present invention may also be used inconjunction with both fresh-frozen and formalin-fixed,paraffin-embedded, or otherwise fixed, tissue blocks prepared for studyby immunohistochemisty.

d. Immunoprecipitation

The fusion proteins of the present invention are particularly useful forthe isolation of CD16 and CD32 by immunoprecipitation.Immunoprecipitation involves the separation of the target antigencomponent from a complex mixture, and is used to discriminate or isolateminute amounts of protein. For the isolation of membrane proteins, cellsmust be solubilized into detergent micelles. Nonionic salts arepreferred, since other agents such as bile salts, precipitate at acid pHor in the presence of bivalent cations.

F. AUTOIMMUNE DISEASES

Autoimmune diseases are processes in which the immune system mounts anattack against body tissue components. This attack may be mediated byanti-tissue component antibodies produced by B lymphocytes or bycell-mediated tissue destructive processes mediated by T cells, by NKcells, and by monocytes/macrophages. In some autoimmune diseases severaltissue damaging mechanisms may operate either concurrently orsequentially. The fusion proteins of the current invention can be usedin the treatment of autoimmune diseases. They can be used to alterimmunity and to deliver therapeutic agents to a delivery site in apatient where the therapeutic agent is effective.

The number of autoimmune diseases is considerable and some persons mayhave more than one autoimmune disease. Similarly, signs and symptoms maycover a wide spectrum and severity may also vary widely betweenafflicted individuals and over time. The reasons why some personsdevelop autoimmunity while others do not are imperfectly understood butcertain recurring themes can be signaled. In many autoimmune processesthere is a genetically determined propensity to develop disease. Amongthe genes that have been linked to propensity to develop autoimmunityare those of the major histocompatibility complex. In addition,environmental factors are thought to play a role. During embryonicdevelopment many of those immune system cells that are capable ofreacting against self-components are eliminated but some remain so thatessentially everyone is at least theoretically capable of mounting anautoimmune response. This observation implies that under normalcircumstances potentially auto-aggressive cells are held in check byphysiologic restraint mechanisms and that a contributor to thepathogenesis of autoimmunity is a failure of normal restraintmechanisms.

Examples of commonly encountered autoimmune disorders include but arenot limited to: systemic lupus erythematosus, rheumatoid arthritis, type1 diabetes, Guillain-Barŕe syndrome, other immune mediated neuropathiesincluding chronic inflammatory demyelinating polyneuropathy, multiplesclerosis and other immune-mediated central nervous system demyelinatingdiseases, rheumatoid arthritis, Crohn's disease, ulcerative colitis,myasthenia gravis, scleroderma/systemic sclerosis, anddermatomyositis/polymyositis to name some of the more commonlyencountered entities. Additional autoimmune diseases include acuteglomerulonephritis, nephrotic syndrome, and idiopathic IgA nephropathyamong autoimmune processes that affect the kidneys.

Examples of autoimmune processes that affect the formed elements of theblood are autoimmune aplastic anemia, autoimmune hemolytic anemia, andidiopathic thrombocytopenic purpura.

Autoimmune diseases that affect the endocrine organs include Addison'sdisease, idiopathic hypoparathyroidism, Grave's disease, Hashimoto'sthyroiditis, lymphocytic hypophysitis, autoimmune oophoritis, andimmunologic infertility in the male.

The liver may also be the target of autoimmune processes. Examplesinclude autoimmune hepatitis, hepatitis C virus-associated autoimmunity,immunoallergic reaction drug-induced hepatitis, primary biliarycirrhosis, and primary sclerosing cholangitis.

Autoimmune processes of the intestinal tract include pernicious anemia,autoimmune gastritis, celiac disease, Crohn's disease, and ulcerativecolitis.

Cutaneous autoimmune diseases include dermatitis herpetiformis,epidermolysis bullosa acquisita, alopecia totalis, alopecia areata,vitiligo, linear IgA dermatosis, pemphigus, pemphigoid, psoriasis,herpes gestationis, and cutaneous lupus including neonatal lupuserythematosus.

Additional autoimmune diseases with rheumatological features includeCREST syndrome, ankylosing spondylitis, Bechet's disease, juvenilerheumatoid arthritis, Sjögren's syndrome, and eosinophilia-myalgiasyndrome.

Autoimmune diseases can affect the heart. Examples include myocarditisand idiopathic dilated cardiomyopathy, rheumatic fever, Chaga's diseaseand possibly some components of atherosclerosis.

There can be an autoimmune component to inflammatory diseases of theblood vessels. Examples include giant cell arteritis, Kawasaki'sdisease, Henoch-Schonlein purpura, polyarteritis nodosa, Goodpasture'ssyndrome, immune complex vasculitis, Wegener's granulomatosis,Churg-Strauss syndrome, Takayasu arteritis, necrotizing vasculitis, andanti-phospholipid antibody syndrome.

Autoimmune diseases of the central and peripheral nervous systems canoccur as a remote effect of malignant tumors. Rarely these same entitiesoccur in the absence of a tumor. Examples include the Lambert-Eatonsyndrome, paraneoplastic myelopathy, paraneoplastic cerebellardegeneration, limbic encephalitis, opsoclonus myoclonus, stiff mansyndrome, paraneoplastic sensory neuropathy, the POEMS syndrome, dorsalroot ganglionitis, and acute panautonomic neuropathy.

Autoimmune diseases may affect the visual system. Examples includeMooren's ulcer, uveitis, and Vogt-Koyanagi-Harada syndrome.

Other autoimmune processes, or ones in which autoimmunity may contributeto disability, include interstitial cystitis, diabetes insipidus,relapsing polychondritis, urticaria, reflex sympathetic dystrophy, andcochleolabyrinthitis.

The list of autoimmune processes given above, while extensive, is notintended to be exhaustive. Rather it is intended to document thatautoimmunity is a wide-ranging clinical phenomenon. Exemplary diseasesin this field such as systemic lupus erythematosus, multiple sclerosis,the Guillain-Barré syndrome, and autoimmune thrombocytopenic purpura arediscussed in further detail below, with a view to providing someunderstanding of the problems involved in the diagnosis and treatment ofthese debilitating and potentially fatal disorders.

1. Systemic Lupus Erythematosus (SLE)

Multiple organ systems are involved in SLE and the manifestations of theillness are protean. Non-erosive and generally non-deforming arthritisand photosensitive rashes occur cumulatively in more than 75% of caseswhile serositis, central nervous system (CNS) involvement, and renalinvolvement occur in about 50% of cases. Lymphopenia occurs in the greatmajority of unselected cases and is almost invariably present in activedisease. Hemolytic anemia and thrombocytopenia occur in about 50% ofcases. SLE may involve any organ in the body. Commonly affected organsinclude the skin, kidneys, serosal membranes, joints, heart, and theCNS. Pathologically, immune complexes are deposited in the glomeruli ofthe kidneys, and immunoglobulin deposits in the skin at dermal epidermaljunctions are the rule. SLE is characterized by numerous autoantibodiesof varying specificities of which antinuclear antibodies (ANA) arealmost invariably present as are antibodies to native double strandedDNA and to denatured single stranded DNA. Such antibodies are useful indiagnosis. Fibrinoid deposits within blood vessels and on serosalsurfaces are another pathologic feature.

The clinical manifestations of SLE are so varied that a list ofdiagnostic criteria to be fulfilled before a definitive diagnosis of thedisease can be made has been developed. 14 criteria are listed, 4 ormore of which must be satisfied for a diagnosis (Cohen et al. 1971). Thecriteria include facial erythema, discoid lupus rash, Raynaud'sphenomenon, alopecia, photosensitivity, oral nasal or pharyngealulceration, arthritis without deformity, LE cells, false positive testfor syphilis, proteinuria (>3.5 g./day), pleuritis, pericarditis,psychosis, convulsions, hemolytic anemia, leukopenia, andthrombocytopenia.

SLE is thought to be primarily an antibody-mediated disease. ANA and DNAantibodies are directed against nucleoproteins but there may be numerousadditional autoantibodies directed against mitochondria, ribosomes,lysosomes, soluble cytoplasmic constituents, red cells, white cells,platelets, and clotting factors to name but some. Why these antibodiesemerge remains unclear but a major thrust of treatment attempts in SLEis to lower autoantibody titers.

There is no specific treatment for SLE but several drugs are known tofavorably alter the natural history of the disease (Lieberman et al.1988; Steinberg and Steinberg, 1991; Vyse and Walport, 1993; Wilke etal. 1991; Miller, 1992; Lubbe et al. 1983; Silman et al. 1988). Acceptedtreatments include non-steroidal anti-inflammatory drugs (NSAIDs;Kimberly, 1988), analgesics, glucocorticoids, (Lube et al. 1983; Edwardset al., 1987), hydroxychloroquine, azathioprine (Silman et al. 1988),cyclophosphamide (Steinberg and Steinberg, 1991), plaquenil (Wallace,1993) and atabrine. Nonetheless treatment remains less than optimal andthe agents mentioned have numerous potentially deleterious side effects.Ultraviolet light is known to exacerbate symptoms of SLE. For thisreason barrier creams are sometimes prescribed. It has been conceded bythose of skill in the art that little has changed in the management ofSLE in recent years (Venables, 1993). The present invention offers theprospect of better management of this disease.

The treatment and study of Ab regulation in SLE with immunoglobulinfusion proteins is contemplated in this invention. The fusion proteinsof the instant invention offer several potential advantages for thestudy of Ab regulation in SLE. They more closely approximate thephysiologic situation in vivo than cell activation via FcγR Abs such asanti-CD16. Previously, functional studies of the low affinity FcγR haveutilized mAb directed at CD16, mAb directed against CD2 which uses thesame pathway in NK cells as CD16, or heat aggregated Ig. Though thesereagents activate the FcγR, there are limitations in their applicationto the study of receptor function. Anti-CD16 mAbs bind specific epitopesof CD16 and crosslink the receptor but there are problems with mAbs forlong term therapy. Abs to CD16 exert different effects on NK cellfunction depending on their binding site. This suggests that theirmechanism of action is far from physiologic. mAb bind small regions ofthe receptor while the natural ligand binds numerous sites on thereceptor that act coordinately to regulate receptor function.

2. Multiple Sclerosis

This disease is characterized by destruction of CNS myelin and of theaxons which myelin ensheathes. The illness most commonly begins withfocal attacks of tissue destruction in the white matter of the CNS whichcause loss of neuronal function and as one attack follows anotherprogressively accumulating disability. After a time most multiplesclerosis patients experience a decline in the frequency of theirattacks but this decline is accompanied by a shift in the naturalhistory of the illness to a slow but inexorable worsening of theirneurological disabilities. The switch from a relapsing-remitting courseto a progressive one ultimately occurs in better than 80% of multiplesclerosis victims.

Multiple sclerosis is an inflammatory disease. Lymphocytes andmacrophages move from the blood into the CNS and attack and destroymyelin and ultimately the myelin forming cells known asoligodendrocytes. The process is one of autoimmunity but the precisetarget within the CNS against which the immune response is directedremains unknown. There is a genetically determined predisposition todevelop multiple sclerosis but there is compelling evidence thatenvironmental factors have a role as well, though the nature of theenvironmental factors in cause remains unknown.

There have been advances in the treatment of multiple sclerosis inrecent years. Three agents are approved for the treatment of MS. Theseare interferon beta1a, interferon beta1b, and glatiramer acetate. Allthree modulate immune responses in a manner that favorably alters thehitherto bleak natural history of MS. Unfortunately all three are onlymodestly effective and each has side effects that are often troublesome.The present invention offers the prospect of a more efficient andeffective therapy for MS.

Experimental autoimmune encephalomyelitis (EAE) is a widely used animalmodel for MS and serves as a useful model for the study of autoimmunediseases. EAE is a disease of the central nervous system and may beinduced in susceptible animals by immunization with neuroantigens. EAEmay also be adoptively transferred from one animal to the next by theserial transfer of T cells reactive against encephalitogenicdeterminants of myelin proteins or by the injection of T cell clonesreactive against encephalitogenic determinants of myelin proteins.Myelin proteins that may be targets of the autoreactive response includeproteolipid apoprotein (PLP), myelin basic protein (MBP), and myelinoligodendrocyte protein (MOG). Depending on the type and strain ofanimal used, the mode of induction, and the neuroantigen administered,the disease may be acute and monophasic in nature, or alternativelychronic, or relapsing-remitting.

Affected animals develop flaccid tails, paralysis of the hindlimbs, andincontinence. In severe disease, movement of the forelimbs may alsobecome impaired and animals may become moribund. Histological analysisof the CNS reveals an inflammatory cell infiltrate during the acutestages of disease which may be accompanied by demyelination of theneurons during chronic phases of the disease. EAE is widely used for thestudy of autoimmune disease and serves as a model for testing potentialefficacy of experimental drugs for the treatment of MS and for thetreatment of autoimmune diseases in general.

The proteins of the current invention were tested for their effect ondisease activity in a mouse model of EAE to gain insight into theirpotential use as therapeutics for the treatment of MS and otherautoimmune diseases. As shown in Example 18, products of the currentinvention inhibited EAE in the SJL/J mouse. Administration of constructHSAR0 and in particular of HSAR4 decreased clinical disease activityduring the early acute stages of disease and decreased the frequency ofand severity of relapses at later time points as compared tosaline-treated controls. Decreased inflammatory cell infiltrates wereobserved in the CNS of construct-treated animals compared to salinetreated-controls.

3. Guillain-Barré Syndrome

The Guillain-Barré syndrome (GBS) comprises a group of autoimmuneneuropathies of subacute onset in which the motor function of peripheralnerves is lost to a variable degree ranging from barely detectableweakness to total motor paralysis requiring ventilatory support. Themost common form of the GBS syndrome in the occident follows, in mostinstances, an infectious illness that is usually respiratory orgastrointestinal. The peripheral nerves are invaded by T cells andmacrophages which attack and destroy the myelin that ensheathes thenerve fibers. Loss of myelin impedes nerve impulse conduction and thiscauses weakness and, in extreme instances, paralysis. The process isordinarily self-limited and myelin loss is followed by repair withrestoration of function to variable degree. Treatment of the GBSsyndrome is imperfect. Some benefit is obtained from plasmapheresis orthe intravenous infusion of immunoglobulin but morbidity remainsconsiderable and there is a need for better treatments. The presentinvention offers the prospect for improved therapy of the GBS syndrome.

A second form of the GBS syndrome is also recognized. This form occursprimarily in the orient. In this form motor weakness or paralysis areagain seen but the autoimmune process is mediated by autoantibodiesdirected against glycolipids expressed on the surface of the nervefibers themselves. In this form, as in the more commonly encounteredform, favorable response to plasmapheresis or to intravenouslyadministered immunoglobulin is sometimes seen.

4. Autoimmune (Idiopathic) Thrombocytopenic Purpura (ATP)

In this autoimmune disease platelets are destroyed by autoantibodiesdirected against antigens present on the individual's own plateletmembrane. The disease may present as an acute process or as a chronicone. The acute process primarily affects young children without a sexpreference. The chronic process usually affects adults in the third tofifth decades and shows a 3:1 female preponderance. Common clinicalfeatures observed when the platelet count falls below 10,000 includepetechiae, purpura, gingival bleeding, epistaxis, and menorrhagia.Autoantibodies directed against glycoproteins expressed on the surfaceof platelets and their predecessors are demonstrable in the majority ofpatients. Platelets with surface bound IgG are largely cleared in thespleen by phagocytic macrophages that recognize damaged platelets viabinding of IgG to the FcγRIIIa receptor expressed on macrophages.Resolution of thrombocytopenia has been reported following infusion ofmonoclonal antibody directed against the FcγRIIIa receptor (Clarkson etal. 1986). NK cell activity is reported as decreased in autoimmunethrombocytopenic purpura (Semple et al. 1991). The present inventionbinds to the FcγRIIIa receptor and activates NK cells and accordinglyoffers the prospect of more effective treatment for this autoimmunedisease. Currently accepted treatments for ATP include glucocorticoids,and intravenous immunoglobulin, and when these fail to control thedisease, as is unfortunately often the case, immunosuppresive andcytoxic agents may have to be administered despite their risks.

5. Diseases Favorably Effected by IgG Therapy

The inventors contemplate the therapeutic use of the fusion proteins ofthe current invention in any disease in which intravenous immunoglobulinhas been previously used. Intravenous immunoglobulin has FDA approvalfor the treatment of ATP. The agent has been proven to be efficacious,based on double-blind controlled trials, in the treatment of theGuillain-Barré syndrome, myasthenia gravis and dermatomyositis.Intravenous immunoglobulin has been reported to be beneficial in morethan 30 immunological diseases. The mechanisms for the beneficialeffects in these diseases are currently unknown but may be mediated byvarious immunomodulating properties of intravenous immunoglobulin(Asghar et al., 1996; Dwyer et al., 1992; Geha et al, 1996; Yu et al.,1999). Some diseases in which immunoglobulins have been used fortreatment are shown in Table 1(http://www.bioscience.org/2000/v5/e/asghar/fulltext.htm).

TABLE 1 Diseases in which beneficial effects of intravenousimmunoglobulin have been demonstrated in small numbers (or groups) ofpatients Disease References Anemias of different types Bjorkholm M.:Intravenous immunoglobulin treatment in cytopenic haematologicaldisorders. J. Intern. Medicine. 234, 119-26 (1993).; Ballester O. F., H.I. Saba, L. C. Moscinski, R. Nelson & P. Foulis P: Pure red cellaplasia: treatment with intravenous immunoglobulin concentrate. Semin.Hematol. 29 (Suppl 2), 106-8 (1992) Neutropenias of different BjorkholmM.: Intravenous immunoglobulin treatment in types cytopenichaematological disorders. J. Intern. Medicine. 234, 119-26 (1993).;Dunkel I. J. & J. B. Bussel: New developments in the treatment ofneutropenia. Am. J. Dis. Children 147, 994-1000 (1993) Multiplesclerosis Lisak R. P.: Intravenous immunoglobulin in multiple sclerosis.Neurology 51 (Suppl 5), S25-29 (1998) Sjögren's syndrome Dupond JL., H.Gil, B. de Wazieres: Five-year efficacy of intravenous gamma globulin totreat dysautonomia in Sjogren's syndrome. Am. J. Medicine. 106, 125,1999; Durez P., L. Tourne, W. Feremans, F. Mascart-Lemone, M. Heenen &T. Appelboom: Dramatic response to intravenous high dose gamma-globulinin refractory vasculitis of the skin associated with Sjogren's syndrome.J. Rheumatology 25, 1032-1033, 1998 Cystic fibrosis Rubin B. K: Emergingtherapies for cystic fibrosis lung disease. Chest 115, 1120-6 (1999)Thyroid related eye disease Baschieri L., A. Antonelli, S. Nardi, B.Alberti, A. Lepri, R. Canapicchi & P. Fallahi: Intravenousimmunoglobulin versus corticosteroid in treatment of Graves'ophthalmopathy. Thyroid 7, 579-85, (1997) Uveitis Rosebaum J. T., R. K.George & C. Gordon: The treatment of refractory uveitis with intravenousimmunoglobulin. Am. J. Ophthalmol. 127, 545-9 (1999) Asthma Kon O. M &N. Barnes. Immunosuppressive treatment in asthma. Br. J. Hospital. Med.57, 383-386 (1997); Balfour-Lynn I.: Difficult asthma. Beyond theguidelines. Arch. Dis, Childhood 80, 201-206 (1999) Ulcerative andCrohn's Levine D. S., S. H. Fischer, D. L. Christie, R. C. Haggitt &disease H. D. Ochs: intravenous immunoglobulin therapy for active,extensive, and medically refractory idiopathic ulcerative or Crohn'scolitis. Am. J. Gastroenterol. 87, 91-100 (1992) Pyoderma gangrenosumGupta A. K., N. H. Shear & D. N. Sauder: Efficacy of human intravenousimmune globulin in pyoderma gangrenosum. J. Am. Acad. Dermatol. 32,140-142 (1995)

G. BIOLOGICAL FUNCTIONAL EQUIVALENTS

As modifications and/or changes may be made in the structure of thepolynucleotides and and/or proteins of the present invention, whileobtaining molecules having similar or improved characteristics, suchbiologically functional equivalents are also encompassed within thepresent invention.

1. Modified Polynucleotides and Polypeptides

The biological functional equivalent may comprise a polynucleotide thathas been engineered to contain distinct sequences while at the same timeretaining the capacity to encode the “wild-type” or standard protein.This can be accomplished owing to the degeneracy of the genetic code,i.e., the presence of multiple codons, which encode for the same aminoacids. In one example, one of skill in the art may wish to introduce arestriction enzyme recognition sequence into a polynucleotide while notdisturbing the ability of that polynucleotide to encode a protein.

In another example, a polynucleotide can be engineered to containcertain sequences that result in (and encode) a biological functionalequivalent with more significant changes. Certain amino acids may besubstituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, antigen-binding regions of antibodies, binding sites onsubstrate molecules, receptors, and such like. So-called “conservative”changes do not disrupt the biological activity of the protein, as thestructural change is not one that impinges on the protein's ability tocarry out its designated function. It is thus contemplated by theinventors that various changes may be made in the sequence of genes andproteins disclosed herein, while still fulfilling the goals of thepresent invention.

In terms of functional equivalents, it is well understood by the skilledartisan that, inherent in the definition of a “biologically functionalequivalent” protein and/or polynucleotide, is the concept that there isa limit to the number of changes that may be made within a definedportion of the molecule while retaining a molecule with an acceptablelevel of equivalent biological activity, such as binding to FcγRs.Biologically functional equivalents are thus defined herein as thoseproteins (and polynucleotides) in which selected amino acids (or codons)may be substituted.

In general, the shorter the length of the molecule, the fewer thechanges that can be made within the molecule while retaining function.Longer domains may have an intermediate number of changes. Thefull-length protein will have the most tolerance for a larger number ofchanges. However, it must be appreciated that certain molecules ordomains that are highly dependent upon their structure may toleratelittle or no modification.

Amino acid substitutions are generally based on the relative similarityof the amino acid side-chain substituents, for example, theirhydrophobicity, hydrophilicity, charge, size, and/or the like. Ananalysis of the size, shape and/or type of the amino acid side-chainsubstituents reveals that arginine, lysine and/or histidine are allpositively charged residues; that alanine, glycine and/or serine are allof similar size; and/or that phenylalanine, tryptophan and/or tyrosineall have a generally similar shape. Therefore, based upon theseconsiderations, arginine, lysine and/or histidine; alanine, glycineand/or serine; and/or phenylalanine, tryptophan and/or tyrosine; aredefined herein as biologically functional equivalents.

To effect more quantitative changes, the hydropathic index of aminoacids may be considered. Each amino acid has been assigned a hydropathicindex on the basis of their hydrophobicity and/or chargecharacteristics, these are: isoleucine (+4.5); valine (+4.2); Leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and/or arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte & Doolittle, 1982, incorporated herein by reference). Itis known that certain amino acids may be substituted for other aminoacids having a similar hydropathic index and/or score and/or stillretain a similar biological activity. In making changes based upon thehydropathic index, the substitution of amino acids whose hydropathicindices are within ±2 is preferred, those which are within ±1 areparticularly preferred, and/or those within ±0.5 are even moreparticularly preferred.

It also is understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biological functional equivalent protein and/orpeptide thereby created is intended for use in immunologicalembodiments, as in certain embodiments of the present invention. U.S.Pat. No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with itsimmunogenicity and/or antigenicity, i.e., with a biological property ofthe protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). In makingchanges based upon similar hydrophilicity values, the substitution ofamino acids whose hydrophilicity values are within ±2 is preferred,those which are within ±1 are particularly preferred, and/or thosewithin ±0.5 are even more particularly preferred.

2. Codons

While discussion has focused on functionally equivalent polypeptidesarising from amino acid changes, it will be appreciated that thesechanges may be effected by alteration of the encoding DNA; taking intoconsideration also that the genetic code is degenerate and that two ormore codons may code for the same amino acid. A table of amino acids andtheir codons is presented below for use in such embodiments, as well asfor other uses, such as in the design of probes and primers and thelike.

TABLE 2 CODON TABLE Amino Acids Codons Alanine Ala A GCA GCC GCG GCUCysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu EGAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys KAAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGO CGA CGC CGG CGU Serine Ser SAGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val VGUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

The term “functionally equivalent codon” is used herein to refer tocodons that encode the same amino acid, such as the six codons forarginine or serine, and also refers to codons that encode biologicallyequivalent amino acids (see Codon Table, above).

It will also be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ sequences, and yet still be essentially as setforth in one of the sequences disclosed herein, so long as the sequencemeets the criteria set forth above, including the maintenance ofbiological protein activity where protein expression is concerned. Theaddition of terminal sequences particularly applies to nucleic acidsequences that may, for example, include various non-coding sequencesflanking either of the 5′ or 3′ portions of the coding region or mayinclude various internal sequences, i.e., introns, which are known tooccur within genes.

3. Altered Amino Acids

The present invention, in many aspects, relies on the synthesis ofpeptides and polypeptides in cyto, via transcription and translation ofappropriate polynucleotides. These peptides and polypeptides willinclude the twenty “natural” amino acids, and post-translationalmodifications thereof However, in vitro peptide synthesis permits theuse of modified and/or unusual amino acids. A table of exemplary, butnot limiting, modified and/or unusual amino acids is provided hereinbelow.

TABLE 3 Modified and/or Unusual Amino Acids Abbr. Amino Acid Abbr. AminoAcid Aad 2-Aminoadipic acid EtAsn N-Ethylasparagine BAad 3-Aminoadipicacid Hyl Hydroxylysine BAla beta-alanine, beta-Amino-propionic AHylallo-Hydroxylysine acid Abu 2-Aminobutyric acid 3Hyp 3-Hydroxyproline4Abu 4-Aminobutyric acid, piperidinic 4Hyp 4-Hydroxyproline acid Acp6-Aminocaproic acid Ide Isodesmosine Ahe 2-Aminoheptanoic acid Aileallo-Isoleucine Aib 2-Aminoisobutyric acid MeGly N-Methylglycine,sarcosine BAib 3-Aminoisobutyric acid Melle N-Methylisoleucine Apm2-Aminopimelic acid MeLys 6-N-Methyllysine Dbu 2,4-Diaminobutyric acidMeVal N-Methylvaline Des Desmosine Nva Norvaline Dpm 2,2′-Diaminopimelicacid Nle Norleucine Dpr 2,3-Diaminopropionic acid Orn Ornithine EtGlyN-Ethylglycine

4. Mimetics

In addition to the biological functional equivalents discussed above,the present inventors also contemplate that structurally similarcompounds may be formulated to mimic the key portions of peptide orpolypeptides of the present invention. Such compounds, which may betermed peptidomimetics, may be used in the same manner as the peptidesof the invention and, hence, also are functional equivalents.

Certain mimetics that mimic elements of protein secondary and tertiarystructure are described in Johnson et al. (1993). The underlyingrationale behind the use of peptide mimetics is that the peptidebackbone of proteins exists chiefly to orient amino acid side chains insuch a way as to facilitate molecular interactions, such as those ofantibody and/or antigen. A peptide mimetic is thus designed to permitmolecular interactions similar to the natural molecule.

Some successful applications of the peptide mimetic concept have focusedon mimetics of β-turns within proteins, which are known to be highlyantigenic. Likely β-turn structure within a polypeptide can be predictedby computer-based algorithms. Once the component amino acids of the turnare determined, mimetics can be constructed to achieve a similar spatialorientation of the essential elements of the amino acid side chains.

Other approaches have focused on the use of small,multidisulfide-containing proteins as attractive structural templatesfor producing biologically active conformations that mimic the bindingsites of large proteins (Vita et al., 1998). A structural motif thatappears to be evolutionarily conserved in certain toxins is small (30-40amino acids), stable, and highly permissive for mutation. This motif iscomposed of a beta sheet and an alpha helix bridged in the interior coreby three disulfides.

Beta II turns have been mimicked successfully using cyclicL-pentapeptides and those with D-amino acids. (Weisshoff et al., 1999).Also, Johannesson et al. (1999) report on bicyclic tripeptides withreverse turn-inducing properties.

Methods for generating specific structures have been disclosed in theart. For example, alpha-helix mimetics are disclosed in U.S. Pat. Nos.5,446,128; 5,710,245; 5,840,833; and 5,859,184. Theses structures renderthe peptide or protein more thermally stable, also increase resistanceto proteolytic degradation. Six, seven, eleven, twelve, thirteen andfourteen membered ring structures are disclosed.

Methods for generating conformationally restricted beta turns and betabulges are described, for example, in U.S. Pat. Nos. 5,440,013;5,618,914; and 5,670,155. Beta-turns permit changed side substituentswithout having changes in corresponding backbone conformation, and haveappropriate termini for incorporation into peptides by standardsynthesis procedures. Other types of mimetic turns include reverse andgamma turns. Reverse turn mimetics are disclosed in U.S. Pat. Nos.5,475,085 and 5,929,237, and gamma turn mimetics are described in U.S.Pat. Nos. 5,672,681 and 5,674,976.

H. PROTEINACEOUS COMPOSITIONS

In certain embodiments, the present invention concerns novelcompositions comprising at least one proteinaceous molecule, such as afusion protein with multiple HCH2 regions. As used herein, a“proteinaceous molecule”, “proteinaceous composition”, “proteinaceouscompound”, “proteinaceous chain” or “proteinaceous material” generallyrefers to, but is not limited to, a protein of greater than about 200amino acids or the full length endogenous sequence translated from agene; a polypeptide of greater than about 100 amino acids; and/or apeptide of from about 3 to about 100 amino acids. All the“proteinaceous” terms described above may be used interchangeablyherein.

In certain embodiments the size of at least one proteinaceous moleculemay comprise, but is not limited to, about 1, about 2, about 3, about 4,about 5, about 6, about 7, about 8, about 9, about 10, about 11, about12, about 13, about 14, about 15, about 16, about 17, about 18, about19, about 20, about 21, about 22, about 23, about 24, about 25, about26, about 27, about 28, about 29, about 30, about 31, about 32, about33, about 34, about 35, about 36, about 37, about 38, about 39, about40, about 41, about 42, about 43, about 44, about 45, about 46, about47, about 48, about 49, about 50, about 51, about 52, about 53, about54, about 55, about 56, about 57, about 58, about 59, about 60, about61, about 62, about 63, about 64, about 65, about 66, about 67, about68, about 69, about 70, about 71, about 72, about 73, about 74, about75, about 76, about 77, about 78, about 79, about 80, about 81, about82, about 83, about 84, about 85, about 86, about 87, about 88, about89, about 90, about 91, about 92, about 93, about 94, about 95, about96, about 97, about 98, about 99, about 100, about 110, about 120, about130, about 140, about 150, about 160, about 170, about 180, about 190,about 200, about 210, about 220, about 230, about 240, about 250, about275, about 300, about 325, about 350, about 375, about 400, about 425,about 450, about 475, about 500, about 525, about 550, about 575, about600, about 625, about 650, about 675, about 700, about 725, about 750,about 775, about 800, about 825, about 850, about 875, about 900, about925, about 950, about 975, about 1000, about 1100, about 1200, about1300, about 1400, about 1500, about 1750, about 2000, about 2250, about2500 or greater amino molecule residues, and any range derivabletherein.

As used herein, an “amino molecule” refers to any amino acid, amino acidderivative, or amino acid mimic as would be known to one of ordinaryskill in the art. In certain embodiments, the residues of theproteinaceous molecule are sequential, without any non-amino moleculeinterrupting the sequence of amino molecule residues. In otherembodiments, the sequence may comprise one or more non-amino moleculemoieties. In particular embodiments, the sequence of residues of theproteinaceous molecule may be interrupted by one or more non-aminomolecule moieties.

Accordingly, the term “proteinaceous composition” encompasses aminomolecule sequences comprising at least one of the 20 common amino acidsin naturally synthesized proteins, or at least one modified or unusualamino acid, including but not limited to those shown in Table 3.

In certain embodiments the proteinaceous composition comprises at leastone protein, polypeptide or peptide. In further embodiments theproteinaceous composition comprises a biocompatible protein, polypeptideor peptide. As used herein, the term “biocompatible” refers to asubstance which produces no significant untoward effects when appliedto, or administered to, a given organism according to the methods andamounts described herein Organisms include, but are not limited to, abovine, a reptilian, an amphibian, a piscine, a rodent, an avian, acanine, a feline, a fungus, a plant, an archebacteria, or a prokaryoticorganism, with a selected animal or human subject being preferred. Suchuntoward or undesirable effects are those such as significant toxicityor adverse immunological reactions. In preferred embodiments,biocompatible protein, polypeptide or peptide containing compositionswill generally be mammalian proteins or peptides, or synthetic proteinsor peptides, each essentially free from toxins, pathogens and harmfulimmunogens.

Proteinaceous compositions may be made by any technique known to thoseof skill in the art, including the expression of proteins, polypeptidesor peptides through standard molecular biological techniques, theisolation of proteinaceous compounds from natural sources, or thechemical synthesis of proteinaceous materials. The nucleotide andprotein, polypeptide and peptide sequences for various genes have beenpreviously disclosed, and may be found at computerized databases knownto those of ordinary skill in the art. One such database is the NationalCenter for Biotechnology Information's Genbank and GenPept databases(http://www.ncbi.nlm.nih.gov/). The coding regions for these known genesmay be amplified and/or expressed using the techniques disclosed hereinor as would be known to those of ordinary skill in the art.Alternatively, various commercial preparations of proteins, polypeptidesand peptides are known to those of skill in the art.

In certain embodiments a proteinaceous compound may be purified.Generally, “purified” will refer to a specific protein, polypeptide, orpeptide composition that has been subjected to fractionation to removevarious other proteins, polypeptides, or peptides, and which compositionsubstantially retains its activity, as may be assessed, for example, bythe protein assays, as would be known to one of ordinary skill in theart for the specific or desired protein, polypeptide or peptide.

In certain embodiments, the proteinaceous composition may comprise atleast one antibody. As used herein, the teen “antibody” is intended torefer broadly to any immunologic binding agent such as IgG, IgM, IgA,IgD and IgE. Generally, IgG and/or IgM are preferred because they arethe most common antibodies in the physiological situation and becausethey are most easily made in a laboratory setting.

Polypeptide regions of proteinaceous compounds may be linked via alinker group. A linker group is able to join the compound of interestvia a biologically-releasable bond, such as a selectively-cleavablelinker or amino acid sequence.

The term “antibody” is used to refer to any antibody-like molecule thathas an antigen binding region, and includes antibody fragments such asFab′, Fab, F(ab′)₂, single domain antibodies (DABs), Fv, scFv (singlechain Fv), and the like. The techniques for preparing and using variousantibody-based constructs and fragments are well known in the art. Meansfor preparing and characterizing antibodies are also well known in theart (See, e.g., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988; incorporated herein by reference).

It is contemplated that virtually any protein, polypeptide or peptidecontaining component may be used in the compositions and methodsdisclosed herein. However, it is preferred that the proteinaceousmaterial is biocompatible. Proteins and peptides suitable for use inthis invention may be autologous proteins or peptides, although theinvention is clearly not limited to the use of such autologous proteins.As used herein, the term “autologous protein, polypeptide or peptide”refers to a protein, polypeptide or peptide which is derived from orobtained from an organism. Organisms that may be used include, but arenot limited to, a bovine, a reptilian, an amphibian, a piscine, arodent, an avian, a canine, a feline, a fungus, a plant, or aprokaryotic organism, with a selected animal or human subject beingpreferred. The “autologous protein, polypeptide or peptide” may then beused as a component of a composition intended for application to theselected animal or human subject. Preferably it is biocompatible (i.e.from mammalian origin for mammals, preferably from human origin forhumans, from canine origin for canines, etc.; it is autologous; it isnon-allergenic, and/or it is non-immunogenic).

I. MECHANISMS OF ACTION AND APPLICATIONS

Autoimmune disease often involves both T-cell and B-cell mediatedcomponents that may act dependently or independently of one another,simultaneously or sequentially, resulting in a host-damaging diseaseoften characterized by tissue or cell compromise and a loss of one ormore bodily functions. Fc receptors and proteins of the complementcascade are often intimately associated with the generation of theautoimmune response, the regulation of the ongoing immune response, andthe effector phase of the immune response (i.e. those mechanisms thatlead to tissue or cell destruction or damage). The fusion proteins ofthe current invention, through their ability to bind Fc receptors and/orcomplement, may influence disease outcome by their impact upon one ormore of these areas.

The fusions proteins of the current invention may favorably alterdisease activity by multiple pathways depending on the fusion proteindesign and type of disease treated. Fusion proteins of the currentinvention may be designed to contain; multiple units of HCH2 regions, orportions thereof, able to bind Fe receptors, multiple units of HCH2regions able to bind complement components, or both. It is contemplatedthat the fusion protein design can be modified to maximize potentialbenefits achieved from its use in treating a specific disease and itscomposition may vary from one disease to the next. For example, for thetreatment of some diseases it may be preferable to retain the Fereceptor binding ability of the fusion proteins but exclude or diminishbinding of components of the complement cascade. The obverse may bepreferred for the treatment of other diseases.

The effect of the fusion proteins on disease outcome will depend notonly on whether they contain multiple units able to bind Fc receptors,multiple units able to bind complement components, or both, but also onother protein domains that may be coexpressed in the fusion proteins togive them an additional function, binding capability, or other addedfeature. An additional modification to the fusion proteins of thecurrent invention includes the binding of additional proteins, proteindomains, or peptides to the fusion proteins that give them an additionalfunction, binding capacity, or other added feature. The flexibility inthe fusion protein design enables the inventors to, depending on diseasetype, modify the fusion proteins of the current invention to maximizetheir therapeutic potential. It is an embodiment of the currentinvention that in addition to the treatment of autoimmunity,modifications of the fusion proteins as described above are applicableto their use in the treatment of neoplasms, the treatment of infectionsby viruses or other pathogens, the treatment of warts, and thepurposeful induction of an immune response directed against a particularantigen or antigens.

Fusion proteins able to bind Fc receptors may influence disease outcomethrough multiple mechanisms including but not limited to blocking Fcreceptor accessibility to endogenously produced Ig and immune complexes.Such blockade would be expected to limit self-antigen presentation byantigen presenting cells and to, as a consequence, diminish autoimmuneresponses. Blockade of Fc receptors may also limit or diminish tissueand cell destruction. Tissue and cell destruction in autoimmune diseaseis often mediated by Fc receptor-expressing effector cells (monocytes,neutrophils, macrophages, microglia, NK cells, as well as other celltypes) that bind self-antigen reactive Ig bound to tissue or cells. Forexample, in ATP, the fusion proteins of the current invention couldlimit platelet destruction and clearance by the body by decreasing theiruptake by Kupffer cells in the liver and spleen via Fc receptor-mediatedmechanisms. Similarly fusion proteins might limit demyelination in theCNS in multiple sclerosis or acetylcholine receptor destruction of motorneural endplates in myasthenia gravis by decreasing macrophageaccessibility to Ig bound to self Ag in target tissues. The fusionproteins may favorably alter numerous autoimmune diseases via similarmechanisms.

The fusion proteins of the current invention may modify autoimmunedisease by activating cells through Fc receptors and thereby alteringthe secretion of immunomodulators, the expression of specific cellsurface markers, or the type or magnitude of specific cell functions.Modulation of protein secretion might include the decreased or increasedproduction of interleukins including but not limited to IL-2, IL-4,IL-10, IL-12, IL-18; cytokines including but not limited to TGFβ, TNFα,TNFβ; interferons γ, β, and α; growth factors, and products of thearachidonate cascade. Cellular functions that may be altered includecellular cytotoxicity, cell division, and activation state.

The fusion protein(s) of the current invention may also be used tosuppress or amplify immunity to a specific antigen. Autoimmune diseasemay be treated by inducing tolerance to a specific antigen or bydeviating the autoimmune response to a specific antigen from a harmfulpathogenic one to a less harmful type. For example, in multiplesclerosis the elaboration of type 1 cytokines (IL-12, IL-2) in responseto autoantigen is generally thought to be deleterious to the host whileinduction of a type 2 response (IL-4, IL-10) is thought to beprotective. The purposeful deviation of the immune response from a Th1type to a Th2 type would likely be beneficial in the treatment ofmultiple sclerosis. In contrast, a Th2 type response is thought to beharmful in other autoimmune diseases such as lupus erythematosus, andconsequently the purposeful deviation of the response to autoantigen inthis disease from a Th2 type response to a Th1 type response wouldlikely be beneficial. Thus, modification of the fusion proteins of thecurrent invention would vary depending on the disease type and themechanisms involved.

It is an embodiment of the current invention to coexpress one or moreprotein domains of a specific antigen or bind one or more specificantigens or antigenic determinants to the fusion protein that wouldinduce a protective immune response, deviate a harmful immune responseto a less harmful one, or induce a state of nonresponsiveness to antigen(Lasalle et al., 1994). For example, the inventors contemplate, in thetreatment of multiple sclerosis, to coexpress a neuroantigen peptide inthe fusion protein that induces a protective Th2 type response or anunresponsive state. A nonlimiting list of potential neuroantigens thatmight be used for the treatment of multiple sclerosis includeproteolipid protein, myelin basic protein and myelin oligodendrocyteglycoprotein. Similarly, a T cell receptor or Ig domain may be expressedin the fusion protein that would induce a protective anti-T cellreceptor or anti-idiotype response. The inventors contemplate thatvarying the protein coexpressed based upon disease type should allow thefusion protein of the current invention to be used for the treatment ofnumerous autoimmune diseases.

As mentioned earlier, the adaptive immune system is often referred to ashaving two components, cellular immunity (or Th1 type response) andhumoral immunity (or Th2 type response). Response to an antigen evokesone or both of these components. Immunomodulators such as lymphokinesand monokines that promote one component often inhibit the other. Thus astrong cellular response will often occur in the presence of a bluntedhumoral response and vice versa. Factors important to the development ofone or the other response include the presence or absence of cytokines,costimulatory factors, as well as other factors that are known to thosefamiliar in the art (Lasalle et al., 1994). For example the presence ofIL-4 has been shown to enhance a Th2 type response while the presence ofinterferon gamma induces a Th1 type response (Swain et al., 1988). Inthe treatment of autoimmune disease, neoplasms, or viral infections, orin the induction of immunity to pathogens by vaccine based therapies, itmay be preferable to selectively modulate one or both of thesecomponents. The coadministration of cytokines, steroids, or otherimmunomodulators may be preferred in the treatment of varying diseasesor when attempting to induce immunity to an antigen or antigens basedupon the type of response desired.

J. RECOMBINANT HCH2 POLYMER CONSTRUCTS IN IMMUNE THERAPY FOR CANCER ANDINFECTION

In another embodiment of the invention, a HCH2 polymer fusion proteincan be constructed to specifically target cells. In one embodiment, thefusion protein of this invention can be constructed to target neoplasticcells, malignant cells that make up the tumor, or cancer cells.Neoplastic cells, cancer cells, or the malignant cells that make up thetumor may be targeted using a ligand or bispecific ligand that has aregion capable of binding to a relatively specific marker of the tumorcell. In a specific embodiment, the fusion protein of this inventionbinds the target cell directly using a Fab′ fragment or Fab′ fragmentscovalently attached to the polymer by genetic or chemical means. Anotheraspect of the invention includes using a HCH2 fusion protein to target adelivery site comprising neoplastic cells for the delivery of a toxin.Toxins kill the neoplastic cells. Another aspect of the inventionincludes using a HCH2 fusion protein to target a delivery sitecomprising neoplastic cells for the delivery of a therapeutic agent.Similarly the HCH2 fusion proteins of the invention can be used totarget cells for the treatment of pathogenic conditions resulting frominfections from bacterial, protozoan, fungal, mycoplasma, rickettsia,and viral agents.

HCH2 polymers are polyvalent resulting in high functional affinity forthe binding to and activation of Fc receptors. Additionally, certainpreparations of HCH2 polymers can bind multiple complement components,thus triggering complement activation cascades. Using alterations to theHCH2 monomer unit that are well known in the art, HCH2 polymers can beproduced for the specific binding to Fc receptors, for the specificbinding to complement factors, or for binding to both Fc receptors andcomplement factors simultaneously. HCH2 polymers fused to mono- orbi-specific Fab fragments or other binding domains described hereinresult in molecules with multispecific binding determinants. Based ontheir ability to engage complement and/or FcR on immune cells as well asto bind one or more antigens expressed on target cells, a particularembodiment of the HCH2 polymers can be used to treat or prevent thereoccurrence of neoplastic diseases including cancer. Similarly the HCH2polymers of the invention can be used to treat pathologic conditionsresulting from infections from bacterial, protozoan, fungal,rickettsial, and viral agents.

HCH2 polymers can direct destruction or removal of target cells by anyone or combination of antibody-mediated cellular effector mechanisms.HCH2 polymers can induce target cell killing through antibody-dependentcell-mediated cytotoxicity (ADCC). ADCC is triggered when HCH2 polymers,bound to target cells, cross-link and aggregate FcγR expressed on immunecells such as NK cells. Once the FcγR on NK cells are engaged, forexample, they trigger a series of events that result in the performmediated lysis of target cell membranes, lysosomal enzyme release andcytotoxic superoxide generation. Activation of FcγR onmonocytes/macrophages, which express all three classes of FcγR, can leadto phagocytosis of intact cells as well as release of soluble cytotoxicfactors. For example, bispecific antibodies that target FcγR and erbB-2protein are known in the art. The erbB-2 protein is over-expressed incancers of the breast, ovary and other cancers. MDX-H210 is a bispecificantibody constructed through the chemical cross-linking of a F(ab′)fragment that recognizes FcγRI (high affinity Fc receptor) to a fragmentthat recognizes the erbB-2 protein. ADCC mediated tumor cell killing ispostulated to proceed as a result of ligation of tumor cell-boundantibody to FcγRI receptors expressed on macrophages (Curnow, 1997).

Complement activation is the other major antibody-mediated pathway forclearance of target cells. HCH2 polymers can induce target cell killingthrough complement-dependent cytotoxicity (CDC). CDC begins with thebinding of C1q to HCH2 regions (Fc regions) bound to target cellantigens. Binding of C1q leads to classical complement pathwayactivation ultimately resulting in the formation of the membrane attackcomplex (factors C5b-9) and disruption of the cell membrane. Classicalpathway activation is not cell-mediated, depending only on circulatingcomplement factors. In addition to CDC, target cell killing can beinduced through complement-dependent cellular cytotoxicity (CDCC). CDCCbegins with the binding of C1q to the complement binding site within theHCH2 region. Other complement factors, such as C3b and C4b, aresubsequently deposited and these trigger enhanced phagocytosis andcytotoxic killing by macrophages, polymorphonulear leukocytes and NKcells. CDC has been identified as a major effector mechanism by whichthe therapeutically administered monoclonal antibody Rituximab exertsits effects in the treatment of low grade non-Hodgkin's lymphoma(Harjunpää et al., 2000).

The fusion proteins of the current invention offer several potentialadvantages for Fc receptor-mediated killing of target cells in additionto those already discussed for monoclonal antibodies or fragments ofmonoclonal antibodies in current use. The number of HCH2 units containedin the polymer region can be altered to hone interaction with FcR and/orwith complement. Secondly, use of the Fc region of the antibody totarget FcR opens up the F(ab)2 region for targeting to two additionalepitopes, resulting in a molecule that is trispecific. The additionalspecificity allows for precise targeting to cells of interest. Thirdly,alteration of the HCH2 unit within the polymer region permits specificengagement of FcR only, complement only, or both. In addition, the HCH2polymer interacts with FcR in a manner identical to that of immunecomplexes. This may provide a qualitatively different signal tocytotoxic effector cells than results from binding of monoclonalantibody to epitopes outside the ligand binding site on FcR.

As described herein, HCH2 fusion proteins can be constructed tospecifically target cells expressing tumor antigens. Neoplastic cells orthe malignant cells that make up a tumor may be targeted using a ligandor bispecific ligand that has a region capable of binding to arelatively specific marker of the tumor cell. The fusion proteins ofthis invention can be constructed to target neoplastic cells. Anotheraspect of the invention includes using HCH2 fusion proteins to target adelivery site comprising neoplastic cells for the delivery of atherapeutic agent.

1. Neoplastic Cell Targets

Many so-called “tumor antigens” have been described, any one of whichcould be employed as a target in connection with the combined aspects ofthe present invention. A large number of exemplary solidtumor-associated antigens are listed herein below. The preparation anduse of antibodies against such antigens is well within the skill of theart, and exemplary antibodies include from gynecological tumor sites: OC125; OC 133; OMI, Mo v1; Mo v2; 3C2; 4C7; ID₃; DU-PAIN-2; F 36/22;4F₇/7A₁₀; OV-TL3; B72.3; DF_(3;) 2C₈/2F₇; MF 116; Mov18; CEA 11-H5; CA19-9 (1116NS 19-9); H17-E2; 791T/36; NDOG_(2;) H317; 4D5, 3H4, 7C2, 6E9,2C4, 7F3, 2H11, 3E8, 5B8, 7D3, SB8; HMFG2; 3.14.A3; from breast tumorsites: DF3; NCRC-11; 3C6F9; MBE6; CLNH5; MAC 40/43; EMA; HMFG1 HFMG2;3.15.C3; M3, M8, M24; M18; 67-D-11; D547Sp, D75P3, H222; Anti-EGF; LR-3;TA1; H59; 10-3D-2; HmAB1,2; MBR 1,2,3; 24•17•1; 24•17•2 (3E1•2);F36/22.M7/105; C11, G3, H7; B6•2; B1•1; Cam 17•1; SM3; SM4; C-Mul (566);4D5 3H4, 7C2, 6E9, 2C4, 7F3, 2H11, 3E8, 5B8, 7D3, 5B8; OC 125; MO v2;DU-PAN-2; 4F₇/7A_(10;) DF₃; B72•3; cccccCEA 11; H17-E2; 3•14•A3; FO23C5;from colorectal tumor sites: B72•3; (17-1A) 1083-17-1A; CO17-1A;ZCE-025; AB2; HT-29-15; 250-30.6; 44X14; A7; GA73•3; 791T/36; 28A32;28.19.8; X MMCO-791; DU-PAN-2; ID_(3;) CEA 11-H5; 2C₈/2F_(7;) CA-19-9(1116NS 19-9); PR5C5; PR4D2; PR4D1; from melanoma sites 4•1; 8•2 M_(17;)96•5; 118•1, 133•2, (113•2); L₁, L₁₀, R₁₀(R₁₉); I_(12;) K_(5;) 6•1; R24;5•1; 225.28S; 465.12S; 9•2•27; F11; 376.96S; 465.12S; 15•75; 15•95;Mel-14; Mel-12; Me3-TB7; 225.28SD; 763.24TS; 705F6; 436910; M148; fromgastrointestinal tumors: ID3; DU-PAN-2; OV-TL3; B72•3; CEA 11-H5;3•14•A3; C COLI; CA-19-9 (1116NS 19-9) and CA50; OC125; from lungtumors: 4D5 3H4, 7C2, 6E9, 2C4, 7F3, 2H11, 3E8, 5B8, 7D3, SB8; MO v2;B72•3; DU-PAN-2; CEA 11-H5; MUC 8-22; MUC 2-63; MUC 2-39; MUC 7-39; andfrom miscellaneous tumors: PAb 240; PAb 246; PAb 1801; ERIC•1; M148;FMH25; 6•1; CA1; 3F8; 4F₇/7A_(10;) 2C₈/2F_(7;) CEA 11-H5.

Another means of defining a targetable tumor is in terms of thecharacteristics of a tumor cell itself, rather than describing thebiochemical properties of an antigen expressed by the cell. Accordingly,the skilled artisan is referred to the ATCC catalogue for the purpose ofexemplifying human tumor cell lines that are publicly available (fromATCC Catalogue). Exemplary cell lines include J82; RT4; ScaBER; T24;TCCSUP; 5637; SK-N-MC; SK-N-SH; SW 1088; SW 1783; U-87 MG; U-118 MG;U-138 MG; U-373 MG; Y79; BT-20; BT-474; MCF7; MDA-MB-134-VI; MDA-MD-157;MDA-MB-175-VII MDA-MB-361; SK-BR-3; C-33 A; HT-3; ME-180; MS751; SiHa;JEG-3; Caco-2; HT-29; SK-CO-1; HuTu 80; A-253; FaDu; A-498; A-704;Caki-1; Caki-2; SK-NEP-1; SW 839; SK-HEP-1; A-427; Calu-1; Calu-3;Calu-6; SK-LU-1; SK-MES-1; SW 900; EB1; EB2; P3HR-1; HT-144; Malme-3M;RPMI-7951; SK-MEL-1; SK-MEL-2; SK-MEL-3; SK-MEL-5; SK-MEL-24; SK-MEL-28;SK-MEL-31; Caov-3; Caov-4; SK-OV-3; SW 626; Capan-1; Capan-2; DU 145;A-204; Saos-2; SK-ES-1; SK-LMS-1; SW 684; SW 872; SW 982; SW 1353; U-2OS; Malme-3; KATO III; Cate-1B; Tera-1; Tera-2; SW579; AN3 CA; HEC-1-A;HEC-1-B; SK-UT-1; SK-UT-1B; SW 954; SW 962; NCI-H69; NCI-H128; BT-483;BT-549; DU4475; HBL-100; Hs 578Bst; Hs 578T; MDA-MB-330; MDA-MB-415;MDA-MB-435S; MDA-MB-436; MDA-MB-453; MDA-MB-468; T-47D; Hs 766T; Hs746T; Hs 695T; Hs 683; Hs 294T; Hs 602; JAR; Hs 445; Hs 700T; H4; Hs696; Hs 913T; Hs 729; FHs 738Lu; FHs 173We; FHs 738B1; NIH:0VCAR-3; Hs67; RD-ES; ChaGo K-1; WERI-Rb-1; NCI-H446; NCI-H209; NCI-H146; NCI-H441;NCI-H82; H9; NCI-H460; NCI-H596; NCI-H676B; NCI-H345; NCI-H820;NCI-H520; NCI-H661; NCI-H510A; D283 Med; Daoy; D341 Med; AML-193 andMV4-11.

One may consult the ATCC Catalogue of any subsequent year to identifyother appropriate cell lines. Also, if a particular cell type isdesired, the means for obtaining such cells, and/or their instantlyavailable source, will be known to those of skill in the particular art.An analysis of the scientific literature will thus readily reveal anappropriate choice of cell for any tumor cell type desired to betargeted.

Recent technological advances enable those familiar in the art torapidly and efficiently compare gene expression in neoplastic tissue tothat of normal tissue. These technological advances include but are notlimited to differential gene analysis using gene chip arrays and proteinarrays. Using these technologies one is able to compare mRNA species andproteins expressed in neoplastic tissue to that found in normal tissue.Those mRNA species or proteins that are differentially expressed inneoplastic tissue compared to normal tissue may be readily discerned.Proteins found to be preferentially expressed in neoplastic tissue or inneoplastic cells using these screening technologies serve as likelycandidates for the further development of cancer or tumor specifictherapies. It is an embodiment of the current invention that tumorassociated proteins or tumor specific proteins discovered using thesetechnologies may be employed as targets in connection with the combinedaspects of the present invention.

2. Anti-Tumor Cell Antibodies

A straightforward means of recognizing a tumor antigen target is throughthe use of an antibody that has binding affinity for the particularantigen. An extensive number of antibodies are known that are directedagainst solid tumor antigens. Certain useful anti-tumor antibodies arelisted above. However, as will be instantly known to those of skill inthe art, certain of the antibodies listed will not have the appropriatebiochemical properties, or may not be of sufficient tumor specificity,to be of use therapeutically. An example is MUC8-22 that recognizes acytoplasmic antigen. Antibodies such as these will generally be of useonly in investigational embodiments, such as in model systems orscreening assays.

Generally speaking, antibodies for use in these aspects of the presentinvention will preferably recognize antigens that are accessible on thecell-surface and that are preferentially, or specifically, expressed bytumor cells. Such antibodies will also preferably exhibit properties ofhigh affinity, such as exhibiting a K_(d) of <200 nM, and preferably, of<100 nM, and will not show significant reactivity with life-sustainingnormal tissues, such as one or more tissues selected from heart, kidney,brain, liver, bone marrow, colon, breast, prostate, thyroid, gallbladder, lung, adrenals, muscle, nerve fibers, pancreas, skin, or otherlife-sustaining organ or tissue in the human body. The “life-sustaining”tissues that are the most important for the purposes of the presentinvention, from the standpoint of low reactivity, include heart, kidney,central and peripheral nervous system tissues and liver. The term“significant reactivity”, as used herein, refers to an antibody orantibody fragment, that, when applied to the particular tissue underconditions suitable for immunohistochemistry, will elicit either nostaining or negligible staining with only a few positive cells scatteredamong a field of negative cells.

Another means of defining a tumor-associated target is in terms of thecharacteristics of the tumor cell, rather than describing thebiochemical properties of an antigen expressed by the cell. Accordingly,the inventors contemplate that any antibody that preferentially binds toa tumor cell may be used as the targeting component of an immunotoxin orcoaguligand. The preferential tumor cell binding is again based upon theantibody exhibiting high affinity for the tumor cell and not havingsignificant reactivity with life-sustaining normal cells or tissues, asdefined above.

To generate a tumor cell-specific antibody, one would immunize an animalwith a composition comprising a tumor cell antigen and, as describedmore fully below, select a resultant antibody with appropriatespecificity. The immunizing composition may contain a purified, orpartially purified, preparation of any of the antigens listed above; acomposition, such as a membrane preparation, enriched for any of theantigens listed above; any of the cells listed above; or a mixture orpopulation of cells that include any of the cell types listed above.

Of course, regardless of the source of the antibody, in the practice ofthe invention in human treatment, one will prefer to ensure in advancethat the clinically-targeted tumor expresses the antigen ultimatelyselected. This is achieved by means of a fairly straightforward assayinvolving antigenically testing a tumor tissue sample, for example, asurgical biopsy, or perhaps testing for circulating shed antigen. Thiscan readily be carried out in an immunological screening assay such asan ELISA (enzyme-linked immunosorbent assay), wherein the bindingaffinities of antibodies from a “bank” of hybridomas are tested forreactivity against the tumor. Antibodies demonstrating appropriate tumorselectivity and affinity are then selected for the preparation ofbispecific antibodies of the present invention.

Due to the well-known phenomenon of cross-reactivity, it is contemplatedthat useful antibodies may result from immunization protocols in whichthe antigens originally employed were derived from an animal, such as amouse or a primate, in addition to those in which the original antigenswere obtained from a human cell. Where antigens of human origin areused, they may be obtained from a human tumor cell line, or may beprepared by obtaining a biological sample from a particular patient inquestion. Indeed, methods for the development of antibodies that are“custom-tailored” to the patient's tumor are known (Stevenson et al.,1990) and are contemplated for use in the fusion proteins of thisinvention.

3. Further Tumor Cell Targets and Binding Ligands

In addition to the use of antibodies, other ligands could be employed todirect a HCH2 fusion protein to a tumor site by binding to a tumor cellantigen. For tumor antigens that are over-expressed receptors (e.g.estrogen receptor, EGF receptor), or mutant receptors, the correspondingligands could be used as targeting agents.

K. HCH2 POLYMERS IN ANTIGEN PRESENTATION TO ANTIGEN PRESENTING CELLS(APC)

In another embodiment of the invention, the polymers of this inventionwill be linked to an antigen. As used herein, the term “antigen” meansany natural or synthetic immunogenic substance, a fragment or portion ofan immunogenic substance, a peptide epitope, or a hapten (as defined inU.S. Pat. No. 5,922,845, p 13). In one embodiment, the polymers of theinvention are used to target an antigen to the cell to enhance theprocess of internalization and presentation of the antigen by thesecells, and ultimately to stimulate an immune response. In anotherembodiment, the polymers of the invention specifically bind the antigendirectly or bind to epitopes attached to the antigen, e.g., a clonedFab′ fragment covalently attached to the polymer by genetic or chemicalmeans which recognizes the antigen or epitopes attached to the antigen,and targets the bound antigen to antigen presenting cells (APC) forinternalization, processing, and presentation. In another embodiment,the antigen is linked to the polymers of the invention and at the sametime binds a surface receptor of an antigen-presenting cell. In apreferred embodiment the antigen is covalently attached to the polymersof the invention by genetic or chemical means.

More broadly, the polymers of this invention will be linked to a cellsurface marker. A cell surface marker is a protein, carbohydrate,glycolipid, etc. but most commonly comprises a protein localized to theplasma membrane of a cell having a portion exposed to the extracellularregion (e.g. an integral membrane protein or a transmembraneglycoprotein), such that the extracellular portion can be specificallybound by an antibody or other ligand. The term cell surface marker alsorefers to a polynucleotide sequence encoding such a cell surfaceprotein. Numerous cell surface proteins can be used as cell surfacemarkers, such as, for example, a CD (cluster of differentiation) antigenpresent on cells of hematopoietic lineage (CD2, CD4, CD8, CD21),Gamma-glutamyltranspeptidase, an adhesion protein (ICAM-1, ICAM-2,ELAM-1, VCAM-1), a hormone, a growth factor, a cytokine receptor, ionchannels, and the membrane-bound form of an immunoglobulin chain.

1. HCH2 Polymers for Use in Vaccines.

Traditional vaccines consist of killed or attenuated pathogenicorganisms or their products administered to develop an immune response.Drawbacks to the traditional approach include unwanted harmful immuneresponses, inoculation with potentially infectious pathogens, and poorimmune responses. Typically these vaccines require co-administration ofpotent adjuvants to elicit effective antibody responses. Vaccines can bemade more effective by delivering those antigenic determinants that aremost likely to confer protective immunity. Early attempts to developpeptide based vaccines resulted in poor immune responses due in part toan inefficient presentation of antigen by APCs.

APCs capture, internalize and present antigen. In addition they provideimportant costimulatory signals to T-cells. T-cells, thus activated, arecapable of stimulating the production of antibody-forming B cells.Monocytes, especially macrophages and dendritic cells, function as APC.Macrophages express all three classes of FcγR constitutively whereasdendritic cells express FcγRI and FcγRII.

Dendritic cells (DCs) are highly specialized and are potent APCs forT-cells. As a result of this capacity DCs are often referred to as‘professional APCs’. DCs present antigen efficiently on both MAC I andMHC II resulting in the initiation of CD8+ and CD4+ responsesrespectively. DCs can prime naive T-cells. Subsequent to activation byDCs, T-cells can interact with other APC. DCs have a proliferativeimmature stage followed by terminal differentiation into anon-proliferative mature stage. Immature DCs express FcγRI and FcγRII,are capable of internalizing and presenting antigen, and synthesizelarge amounts of MHC II. In contrast mature DCs no longer express FcγRs,become fully active APCs, activate T-cells, and secrete large amounts ofIL-12 (which spurs differentiation of T-cells). DCs are a more potentAPC than macrophages though much less numerous.

There has been great interest in the enhancement of antigen presentationby targeting antigen to FcγR expressed on APCs. Known in the art arepeptide vaccines wherein antigenic determinants are grafted into thevariable region of IgG. These ‘antigenized-antibodies’ increased thehalf-life of antigen and facilitated uptake of antigen by APCs via theFcγRI receptor (Zaghouani et al., 1993; Zanetti et al., 1992). Use ofantigenized-antibodies have been shown to be more effective at primingantigen specific T-cell responses than peptide alone.Antigenized-antibodies have several limiting features: Since they aredirected towards FcγRI alone, they can be effectively competed againstby monomeric serum IgG. Secondly, the design of the molecule limits thesize of the antigenizing determinant to a small peptide fragment.

More recently antigen has been expressed as a fusion protein with orchemically conjugated to monoclonal antibodies and Fab fragmentsdirected against FcγRI and FcγRII (Liu et al., 1996b; Guyre et al.,1997). Using tetanus toxoid epitopes conjugated to anti-FcγRI monoclonalantibody, one group found that peptides directed to FcγRI were 100 to1000 fold more efficient than peptide alone in T-cell stimulation (Liuet al., 1996a). However, use of anti-FcγRI Fab′ required chemicalcross-linking to achieve maximal responses to antigen, thus implicatingthe low affinity IgG receptors (Keler et al., 2000). Disadvantages ofthis approach include the promiscuous binding of antigen-linkedmonoclonal antibody to FcγRI expressed on non-APCs. Monoclonalantibodies trigger effector functions poorly. Fab′ fragments have theadditional disadvantage of a short half-life in the circulation.

Attachment of antigen to the HCH2 polymers described herein for thepurpose of targeting APCs has distinct advantages over existingstrategies. HCH2 polymers present antigen to low affinity receptors(FcγRII and FcγRIII), thus bypassing competition from monomeric serumIgG for binding to FcγRI. Additionally there is no need for chemicalcross-linking as is necessary when using anti-FcγRI Fab′. HCH2 polymersimitate immune complexes. Antigen presented in the context of an immunecomplex may be a particularly appropriate substrate for APCs.Antigen-linked HCH2 polymers efficiently trigger effector functions thataugment the immune response.

2. HCH2 Polymers in Tolerance Induction.

Immunologic tolerance is characterized by the selective lack of animmune response, including lack of a pathogenic immune response to aspecific antigen even while leaving other responses of the immune systemintact. Induction and maintenance of T cell unresponsiveness to aspecific antigen may be achieved by several mechanisms that can bebroadly summarized as: 1) clonal deletion; 2) anergy; and 3)suppression. Clonal deletion is a process of negative selection wherebyT cells with high affinity for self-antigens are deleted in the thymus.Deletion is achieved by programmed cell death (apoptosis). This processof negative selection in the thymus is known as ‘central tolerance’.Anergy represents a state of immune inactivation characterized byabolished proliferative and cytokine responses. It is induced in cellsthat previously responded to a given antigen and results in anunresponsive state upon re-stimulation with antigen. Since thismechanism acts upon mature T cells that have exited the thymus andreside in the peripheral compartments, this form of tolerance is termed‘peripheral tolerance’. Anergy is induced by any of a number ofmolecular events and need not be permanent: it can be reversed bycertain cytokines. Three common anergy-inducive mechanisms are T cellreceptor (TCR) stimulation without co-stimulatory signals, sub-optimalTCR stimulation even in the presence of co-stimulation, and theautocrine inhibitory actions of IL-10. Suppression of T cell function isa third mechanism by which T cell tolerance can be achieved. Suppressionensues when regulatory T cells are induced to exert “non-specific”suppressive effects on antigen-specific T cells in their vicinity. Thismicroenvironmental effect is also referred to as ‘bystander suppression’

B cell tolerance involves concepts and mechanisms similar but notidentical to those encountered in T cell tolerance. In mature B cellstolerance can be induced through a block in Ig-receptor signaling whichresults in impaired expression of the B7 costimulatory molecules.

Induction of tolerance to either self- or foreign-antigens provides animportant therapeutic approach to the treatment of allergies, autoimmunedisease and host vs. graft disease (transplant rejection). In addition,the therapeutic potential of many foreign biologically active agents islimited by their immunogenicity. Tolerance induction represents oneapproach for the control of immune responses directed againstbiologically active foreign agents, thus improving their therapeuticpotential. In most instances the antigen to be tolerized is presentedorally, intradermally, or intravenously. The source of antigen can be inthe form of a peptide, a protein, or nucleic acid which can express apeptide or protein. The antigen is then internalized by antigenpresenting cells (APC) and presented on the surface of the cell, mosttypically as a MHC I-antigen complex or as a MHC II-antigen complex.

The HCH2 polymers of the current invention have several advantageousaspects for use as vehicles for tolerance induction. Antigen(s) linkedto the HCH2 polymers by chemical or genetic means are targeted to Fcreceptors expressed on APC such as macrophages, B cells, and dendriticcells (DC). Fc-receptor-mediated internalization results in processingand presentation of antigen at the cell surface—the key first step intolerance induction.

Macrophages and DC express Fc receptors for both IgG and IgE. HCH2polymers are expressible which bind both classes of Fc receptorsimultaneously—coaggregation of different Fc receptor classes may haveadvantages over targeting a single class of receptor. Ligation of FcRsinduces secretion of IL10 from certain immune cells and, as alreadynoted, IL10 induces anergy in T cells. As is observed for immunecomplexes, binding of HCH2 polymers to FcRs may induce a pattern ofcytokine secretion that deviates T cell immune responses from a TH1 typeresponse to a TH2 type response. TH2 type T cells favor theestablishment and maintenance of immune tolerance. Therefore, antigenslinked to the HCH2 polymers can promote tolerance induction by both theefficient presentation of antigen to APC and the simultaneous inductionof mechanisms that favor establishment of immune tolerance.

L. TOXINS

For certain applications, it is envisioned that therapeutic orpharmacological agents will be attached to the HCH2 fusion proteins,particularly cytotoxic or otherwise anti-cellular agents having theability to kill or suppress the growth or cell division of neoplasticcells. In general, an aspect of the invention contemplates the use ofany pharmacological agent that can be conjugated to the HCH2 fusionproteins which has been constructed to target a specific region, anddelivered in active form to the targeted cell. Exemplary anti-cellularagents include chemotherapeutic agents, radioisotopes as well ascytotoxins. In the case of chemotherapeutic agents, agents such as ahormone such as a steroid, an anti-metabolite such as cytosinearabinoside, fluorouracil, methotrexate or aminopterin; ananthracycline; mitomycin C; a vinca alkaloid; demecolcine; etoposide;mithramycin; or an anti-tumor alkylating agent such as chlorambucil ormelphalan, will be particularly preferred. Other embodiments may includeagents such as a cytokine, growth factor, bacterial endotoxin or thelipid A moiety of bacterial endotoxin.

In certain preferred embodiments, the immunotoxins will includegenerally a plant-, fungus- or bacterium-derived toxin, such as an Achain toxin, a ribosome inactivating protein, α-sarcin, aspergillin,restrictocin, a ribonuclease, diphtheria toxin or pseudomonas exotoxin,to mention just a few examples. The use of toxin-protein constructs iswell known in the art of immunotoxins, as is their attachment toantibodies. Of these, a particularly preferred toxin will be adeglycosylated ricin A chain. Deglycosylated ricin A chain is preferredbecause of its extreme potency and long half-life.

1. Preparation of Targeting Agent-Toxin Conjugates

While the preparation of immunotoxins is, in general, well known in theart (see, e.g., patents U.S. Pat. No. 4,340,535, and EP 44167, bothincorporated herein by reference), the inventors are aware that certainadvantages may be achieved through the application of certain preferredtechnology, both in the preparation of the immunotoxins and in theirpurification for subsequent clinical administration

Additionally, while numerous types of disulfide-bond containing linkersare known which can successfully be employed to conjugate the toxinmoiety with the targeting agent, certain linkers will generally bepreferred over other linkers, based on differing pharmacologicalcharacteristics and capabilities. For example, linkers that contain adisulfide bond that is sterically “hindered” are to be preferred, due totheir greater stability in vivo, thus preventing release of the toxinmoiety prior to binding at the site of action. A wide variety ofcytotoxic agents are known that may be conjugated to HCH2 polymers.Examples include numerous useful plant-, fungus- or evenbacterium-derived toxins, which, by way of example, include various Achain toxins, particularly ricin A chain, ribosome inactivating proteinssuch as saporin or gelonin, α-sarcin, aspergillin, restrictocin,ribonucleases such as placental ribonuclease, diphtheria toxin, andpseudomonas exotoxin, to name just a few.

Depending on the specific toxin compound used as part of the fusionprotein, it may be necessary to provide a peptide spacer operativelyattaching the targeting agent and the toxin compound which is capable offolding into a disulfide-bonded loop structure. Proteolytic cleavagewithin the loop would then yield a heterodimeric polypeptide wherein thetargeting agent and the toxin compound are linked by only a singledisulfide bond. See, for example, Lord et al. (1992). An example of sucha toxin is a Ricin A-chain toxin.

When certain other toxin compounds are utilized, a non-cleavable peptidespacer may be provided to operatively attach the targeting agent and thetoxin compound of the fusion protein. Toxins which may be used inconjunction with non-cleavable peptide spacers are those which may,themselves, be converted by proteolytic cleavage, into a cytotoxicdisulfide-bonded form (see for example, Ogata et al., 1990). An exampleof such a toxin compound is a Pseudonomas exotoxin compound.

Nucleic acids that may be utilized herein comprise nucleic acidsequences that encode a targeting agent of interest and nucleic acidsequences that encode a toxin agent of interest. Such targetagent-encoding and toxin agent-encoding nucleic acid sequences areattached in a manner such that translation of the nucleic acid yieldsthe targeting agent/toxin compounds of the invention.

2. Attachment of Other Agents to Targeting Agents

It is contemplated that most therapeutic applications of the additionalimmunotoxin aspects of the present invention will involve the targetingof a toxin moiety to a tumor cell. This is due to the much greaterability of most toxins to deliver a cell killing effect as compared toother potential agents. However, there may be circumstances, such aswhen the target antigen does not internalize by a route consistent withefficient intoxication by targeting agent/toxin compounds, such asimmunotoxins, where one will desire to target chemotherapeutic agentssuch as anti-tumor drugs, other cytokines, antimetabolites, alkylatingagents, hormones, and the like. The advantages of these agents overtheir non-targeting agent conjugated counterparts is the addedselectivity afforded by the targeting agent, such as an HCH2 polymerfusion protein. One might mention by way of example agents such assteroids, cytosine arabinoside, methotrexate, aminopterin,anthracyclines, mitomycin C, vinca alkaloids, demecolcine, etoposide,mithramycin, and the like. This list is, of course, merely exemplary inthat the technology for attaching pharmaceutical agents to targetingagents, such as antibodies or antibody fusion protein, for specificdelivery to tissues is well established (see, e.g., Ghose and Blair,1987).

A variety of chemotherapeutic and other pharmacological agents have nowbeen successfully conjugated to antibodies and shown to functionpharmacologically (see, e.g., Vaickus et al., 1991). Exemplaryantineoplastic agents that have been investigated include doxorubicin,daunomycin, methotrexate, vinblastine, and various others (Dillman etal., 1988; Pietersz et al., 1988). Moreover, the attachment of otheragents such as neocarzinostatin (Kimura et al., 1983), inacromycin(Manabe et al., 1984), trenimon (Ghose, 1982) and α-amanitin (Davis andPreston, 1981) has been described.

M. BISPECIFIC ANTIBODIES

The use of bispecific antibodies (BsAbs) is contemplated in the fusionproteins and the methods for treating disease and targeting deliverysites of the current invention. In general, the preparation of BsAbs isalso well known in the art, as exemplified by Glennie et al. (1987).

BsAbs have also been developed particularly for use as immunotherapeuticagents. As mentioned earlier in conjunction with antigen-induction,certain of these antibodies were developed to cross-link lymphocytes andtumor antigens (Nelson, 1991; Wunderlich, et al., 1992). Examplesinclude chimeric molecules that bind T cells, e.g., at CD3, and tumorantigens, and trigger lymphocyte-activation by physically cross-linkingthe TCR/CD3 complex in close proximity to the target cell (Staerz etal., 1985; Perez et al., 1985; 1986a; 1986b; Ting et al., 1988).

Indeed, tumor cells of carcinomas, lymphomas, leukemias and melanomashave been reported to be susceptible to BsAb-mediated killing by T cells(Nelson, 1991; Segal et al., 1992). These types of BsAbs have also beenused in several Phase I clinical trials against diverse tumor targets.The bispecific cross-linking antibodies may be administered as describedin references such as Kroesen et al. (1997); Bolhuis et al. (1992); andNitta et al. (1990).

While numerous methods are known in the art for the preparation ofBsAbs, the Glennie et al. (1987) method involves the preparation ofpeptic F(ab′γ)₂ fragments from the two chosen antibodies, followed byreduction of each to provide separate Fab′γ_(SH) fragments. The SHgroups on one of the two partners to be coupled are then alkylated witha cross-linking reagent such as o-phenylenedimaleimide to provide freemaleimide groups on one partner. This partner may then be conjugated tothe other by means of a thioether linkage, to give the desired F(ab′γ)₂heteroconjugate.

Another method for producing BsAbs is by the fusion of two hybridomas toform a quadroma (Fanger et al., 1992; Nolan et al, 1990; Menard et al.,1989). As used herein, the term “quadroma” is used to describe theproductive fusion of two B cell hybridomas. Using now standardtechniques, two antibody producing hybridomas are fused to give daughtercells, and those cells that have maintained the expression of both setsof clonotype immunoglobulin genes are then selected.

A preferred method of generating a quadroma involves the selection of anenzyme deficient mutant of at least one of the parental hybridomas. Thisfirst mutant hybridoma cell line is then fused to cells of a secondhybridoma that had been lethally exposed, e.g., to iodoacetamide,precluding its continued survival. Cell fusion allows for the rescue ofthe first hybridoma by acquiring the gene for its enzyme deficiency fromthe lethally treated hybridoma, and the rescue of the second hybridomathrough fusion to the first hybridoma. Preferred, but not required, isthe fusion of immunoglobulins of the same isotype, but of a differentsubclass. A mixed subclass antibody permits the use of an alternativeassay for the isolation of a preferred quadroma.

In more detail, one method of quadroma development and screeninginvolves obtaining a hybridoma line that secretes the first chosen MAband making this deficient for the essential metabolic enzyme,hypoxanthine-guanine phosphoribosyltransferase (HGPRT). To obtaindeficient mutants of the hybridoma, cells are grown in the presence ofincreasing concentrations of 8-azaguanine (1×10⁻⁷M to 1×10⁻⁵M). Themutants are subcloned by limiting dilution and tested for theirhypoxanthine/aminopterin/thymidine (HAT) sensitivity. The culture mediummay consist of, for example, DMEM supplemented with 10% FCS, 2 mML-Glutamine and 1 mM penicillin-streptomycin.

A complementary hybridoma cell line that produces the second desired MAbis used to generate the quadromas by standard cell fusion techniques(Galfre et al., 1981), or by using the protocol described by Clark etal. (1988). Briefly, 4.5×10⁷ HAT-sensitive first cells are mixed with2.8×10⁷ HAT-resistant second cells that have been pre-treated with alethal dose of the irreversible biochemical inhibitor iodoacetamide (5mM in phosphate buffered saline) for 30 minutes on ice before fusion.Cell fusion is induced using polyethylene glycol (PEG) and the cells areplated out in 96 well microculture plates. Quadromas are selected usingHAT-containing medium. BsAb-containing cultures are identified using,for example, a solid phase isotype-specific ELISA and isotype-specificimmunofluorescence staining.

In identification embodiments, ELISA, FACS, immunofluorescence staining,idiotype specific antibodies, antigen binding competition assays, andother methods common in the art of antibody characterization may be usedin conjunction with the present invention to identify preferredquadromas.

Following the isolation of the quadroma, the BsAbs are purified awayfrom other cell products. This may be accomplished by a variety ofprotein isolation procedures, known to those skilled in the art ofimmunoglobulin purification. Means for preparing and characterizingantibodies are well known in the art (See, e.g., Antibodies: ALaboratory Manual, 1988).

For example, supernatants from selected quadromas are passed overprotein A or protein G sepharose columns to bind IgG (depending on theisotype). The bound antibodies are then eluted with, e.g. a pH 3.0citrate buffer. The eluted fractions containing the BsAbs, are dialyzedagainst an isotonic buffer. Alternatively, the eluate is passed over ananti-immunoglobulin-sepharose column. The BsAb is then eluted with 3.5 Mmagnesium chloride. BsAbs purified in this way are then tested forbinding activity by, e.g., an isotype-specific ELISA andimmunofluorescence staining assay of the target cells, as describedabove.

Purified BsAbs and parental antibodies may also be characterized andisolated by SDS-PAGE electrophoresis, followed by staining with silveror Coomassie blue. This is possible when one of the parental antibodieshas a higher molecular weight than the other, wherein the band of theBsAbs migrates midway between that of the two parental antibodies.Reduction of the samples verifies the presence of heavy chains with twodifferent apparent molecular weights.

Furthermore, recombinant technology is now available for the preparationof antibodies in general, allowing the preparation of recombinantantibody genes encoding an antibody having the desired dual specificity.Thus, after selecting the monoclonal antibodies having the mostpreferred binding characteristics, the respective genes for theseantibodies can be isolated, e.g., by immunological screening of a phageexpression library (Oi and Morrison, 1986; Winter and Milstein, 1991;Marks et al., 1991). Then, through rearrangement of Fab coding domains,the appropriate chimeric construct can be readily obtained.

N. COMBINED TREATMENT

Combination of the fusion proteins of the current invention with othertherapeutic agents is contemplated for use in the clinical treatment ofvarious diseases that involve altering immunity, inflammation orneoplasms.

Naturally, before wide-spread use, animal studies and clinical trialswill be conducted. The various elements of conducting a clinical trial,including patient treatment and monitoring, will be known to those ofskill in the art in light of the present disclosure.

The present invention contemplates that the fusion proteins may be usedin combination with other therapies. Therapies for autoimmune diseasesinclude but are not limited to interferon-β, interferon-α, i.v.immunoglobulins, monoclonal antibodies such as h5G1.1-mAb, polyclonalantibodies such as anti-RhoD (WinRho SDF), retinoic acid and otherimmunomodulatory agents such as glatiramer acetate.

Therapies for diseases that involve inflammation include, but are notlimited to non-steroidal inflammatory drugs (NSALDs) such ascyclo-oxygenase 2 (COX-2) inhibitors.

The present invention contemplates that the fusion proteins may be usedas an adjuvant in combination with vaccines. Vaccines include, forexample, mAb 105AD7 anti-idiotype vaccine, mAb 11D10 anti-idiotypevaccine, mAb 3H1 anti-idiotype vaccine, GM2, GM2-KLH, and MUC-1 antigenamong many others.

Cancer therapies include a variety of combination therapies that arecontemplated with the fusion proteins of the current invention includingimmunological, chemical and radiation based treatments. Combinationimmunotherapies include, for example, interleukin-2, monoclonal and/orbispecific antibodies such as Rituximab, Herceptin (Trastuzumab), mAbLym-1, mAb m170, mAb BC8, mAb Anti-B1 (tositumomab), Campath-1H,anti-CEA mAb MN-14, mAb HuG1-M195, mAb HuM291, mAb 3F8, mAb C225(cetuximab), anti-Tac mAb (daclizumab), and mAb hLL2 (epratuzumab).

Combination immunotherapies also include monoclonal antibodies (mAb)linked to toxins or other agents. Examples include mAb gemtuzumabozogamicin (mylotarg), mAb Mono-dgA-RFB4, mAb ibritumomab tiuxetan(IDEC-Y2B8), and Anti-Tac(Fv)-PE38. Combination chemotherapies include,for example, cisplatin (CDDP), carboplatin, procarbazine,mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan,chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin,doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16),tamoxifen, taxol, transplatinum, 5-fluorouracil, vincristin, vinblastinand methotrexate or any analog or derivative variant thereof.

For precancerous conditions such as benign prostatic hyperplasia, asecond therapeutic agent selected from an α-1 adrenergic receptorblocker such as terazosin, doxazosin, prazosin, bunazosin, indoramin,tamsulosin, prazicin or alfuzosin; a 5-α-reductase enzyme blocker suchas finasteride or an azasteroid derivative; a combination of an α-1adrenergic receptor blocker, and a 5-α-reductase enzyme blocker, apotassium channel opener such as minoxidil, and a retinoic acidderivative.

Various combinations may be employed, for instance where the fusionprotein of the current invention is “A” and the radio-, chemotherapeuticor other therapeutic agent is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/AThe terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic composition and achemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing or stasis, both agents are delivered to a cell in acombined amount effective to kill the cell or prevent it from dividing.

The therapy including the fusion protein of the current invention mayprecede or follow the other agent treatment by intervals ranging fromminutes to weeks. In embodiments where the other agent and fusionprotein are applied separately to the cell, one would generally ensurethat a significant period of time did not expire between the time ofeach delivery, such that the agent and the fusion protein would still beable to exert an advantageously combined effect on the cell. In suchinstances, it is contemplated that one would contact the cell with bothmodalities within about 12-24 h of each other and, more preferably,within about 6-12 h of each other, with a delay time of only about 12 hbeing most preferred. In some situations, it may be desirable to extendthe time period for treatment significantly, however, where several days(2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) elapsebetween the respective administrations.

O. PHARMACEUTICAL COMPOSITIONS

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more fusion proteins, therapeutic agents oradditional agent dissolved or dispersed in a pharmaceutically acceptablecarrier. Aqueous compositions of the present invention comprise aneffective amount of the fusion protein, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. The phrases“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, or a human, asappropriate.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drags, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). The use of such media and agents for pharmaceuticalactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active ingredient,its use in the therapeutic compositions is contemplated. Supplementaryactive ingredients can also be incorporated into the compositions. Forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologic Standards.

The biological material should be extensively dialyzed to removeundesired small molecular weight molecules and/or lyophilized for moreready formulation into a desired vehicle, where appropriate. The activecompounds will then generally be formulated for parenteraladministration, e.g., formulated for injection via the intravenous,intramuscular, sub-cutaneous, intranasal, intralesional, or evenintraperitoneal routes. Typically, such compositions can be prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for using to prepare solutions or suspensions upon the additionof a liquid prior to injection can also be prepared; and thepreparations can also be emulsified.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The fusion proteins of the present invention can be formulated into acomposition in a free base, in a neutral or salt form. Pharmaceuticallyacceptable salts, include the acid addition salts (formed with the freeamino groups of the protein) and which are formed with inorganic acidssuch as, for example, hydrochloric or phosphoric acids, or such organicacids as acetic, oxalic, tartaric, mandelic, and the like. Salts formedwith the free carboxyl groups can also be derived from inorganic basessuch as, for example, sodium, potassium, ammonium, calcium, or ferrichydroxides, and such organic bases as isopropylamine, trimethylamine,histidine, procaine and the like.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The preparation of more, or highly, concentratedsolutions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small area.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, intranasal, and intraperitonealadministration. In this connection, sterile aqueous media which can beemployed will be known to those of skill in the art in light of thepresent disclosure. For example, one dosage could be dissolved in 1 mlof isotonic NaCl solution and either added to 1000 ml of hypodermoclysisfluid or injected at the proposed site of infusion, (see for example,“Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and1570-1580). Some variation in dosage will necessarily occur depending onthe condition of the subject being treated. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual subject.

In addition to the compounds formulated for parenteral administration,such as intravenous or intramuscular injection, other pharmaceuticallyacceptable forms include, e.g., tablets or other solids for oraladministration; liposomal formulations; time release capsules; and anyother form currently used, including cremes.

In certain embodiments, the use of liposomes and/or nanoparticles iscontemplated for the formulation and administration of the fusionproteins and/or analogs thereof The formation and use of liposomes isgenerally known to those of skill in the art, and is also describedbelow.

Nanocapsules can generally entrap compounds in a stable and reproducibleway. To avoid side effects due to intracellular polymeric overloading,such ultrafine particles (sized around 0.1 μm) should be designed usingpolymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use in the present invention, and such particles areeasily made.

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core.

The following information may also be utilized in generating liposomalformulations. Phospholipids can form a variety of structures other thanliposomes when dispersed in water, depending on the molar ratio of lipidto water. At low ratios the liposome is the preferred structure. Thephysical characteristics of liposomes depend on pH, ionic strength andthe presence of divalent cations. Liposomes can show low permeability toionic and polar substances, but at elevated temperatures undergo a phasetransition which markedly alters their permeability. The phasetransition involves a change from a closely packed, ordered structure,known as the gel state, to a loosely packed, less-ordered structure,known as the fluid state. This occurs at a characteristicphase-transition temperature and results in an increase in permeabilityto ions, sugars and drugs.

Liposomes interact with cells via four different mechanisms: Endocytosisby phagocytic cells of the reticuloendothelial system such asmacrophages and neutrophils;

adsorption to the cell surface, either by nonspecific weak hydrophobicor electrostatic forces, or by specific interactions with cell-surfacecomponents; fusion with the plasma cell membrane by insertion of thelipid bilayer of the liposome into the plasma membrane, withsimultaneous release of liposomal contents into the cytoplasm; and bytransfer of liposomal lipids to cellular or subcellular membranes, orvice versa, without any association of the liposome contents. Varyingthe liposome formulation can alter which mechanism is operative,although more than one may operate at the same time.

The therapeutic agent may comprise different types of carriers dependingon whether it is to be administered in solid, liquid or aerosol form,and whether it needs to be sterile for such routes of administration asinjection. The present invention can be administered intravenously,intradermally, intraarterially, intraperitoneally, intralesionally,intracranially, intraarticularly, intraprostaticaly, intrapleurally,intratracheally, intranasally, intravitreally, intravaginally,intrarectally, topically, intratumorally, intramuscularly,intraperitoneally, subcutaneously, subconjunctival, intravesicularlly,mucosally, intrapericardially, intraumbilically, intraocularally,orally, topically, locally, by inhalation (e.g. aerosol inhalation), byinjection, by infusion, by continuous infusion, localized perfusionbathing target cells directly, via a catheter, via a lavage, in cremes,in lipid compositions (e.g., liposomes), or by other methods or anycombination of the foregoing as would be known to one of ordinary skillin the art (see, for example, Remington's Pharmaceutical Sciences, 18thEd. Mack Printing Company, 1990, incorporated herein by reference).

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and the route of administration.The practitioner responsible for administration will, in any event,determine the concentration of active ingredient(s) in a composition andappropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, an active compound may comprise between about 2% to about75% of the weight of the unit, or between about 25% to about 60%, forexample, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from about 1 microgram/kg/bodyweight, about 5 microgram/kg/body weight, about 10 microgram/kg/bodyweight, about 50 microgram/kg/body weight, about 100 microgram/kg/bodyweight, about 200 microgram/kg/body weight, about 350 microgram/kg/bodyweight, about 500 microgram/kg/body weight, about 1 milligram/kg/bodyweight, about 5 milligram/kg/body weight, about 10 milligram/kg/bodyweight, about 50 milligram/kg/body weight, about 100 milligram/kg/bodyweight, about 200 milligram/kg/body weight, about 350 milligram/kg/bodyweight, about 500 milligram/kg/body weight, to about 1000 mg/kg/bodyweight or more per administration, and any range derivable therein. Innon-limiting examples of a derivable range from the numbers listedherein, a range of about 5 mg/kg/body weight to about 100 mg/kg/bodyweight, about 5 microgram/kg/body weight to about 500 milligram/kg/bodyweight, etc., can be administered, based on the numbers described above.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof. In many cases,it will be preferable to include isotonic agents, such as, for example,sugars, sodium chloride or combinations thereof.

In other embodiments, one may use eye drops, nasal solutions or sprays,aerosols or inhalants in the present invention. Such compositions aregenerally designed to be compatible with the target tissue type. In anon-limiting example, nasal solutions are usually aqueous solutionsdesigned to be administered to the nasal passages in drops or sprays.Nasal solutions are prepared so that they are similar in many respectsto nasal secretions, so that normal ciliary action is maintained. Thus,in preferred embodiments the aqueous nasal solutions usually areisotonic or slightly buffered to maintain a pH of about 5.5 to about6.5. In addition, antimicrobial preservatives, similar to those used inophthalmic preparations, drugs, or appropriate drug stabilizers, ifrequired, may be included in the formulation. For example, variouscommercial nasal preparations are known and include drugs such asantibiotics or antihistamines.

In certain embodiments the fusion proteins are prepared foradministration by such routes as oral ingestion. In these embodiments,the solid composition may comprise, for example, solutions, suspensions,emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatincapsules), sustained release formulations, buccal compositions, troches,elixirs, suspensions, syrups, wafers, or combinations thereof. Oralcompositions may be incorporated directly with the food of the diet.Preferred carriers for oral administration comprise inert diluents,assimilable edible carriers or combinations thereof. In other aspects ofthe invention, the oral composition may be prepared as a syrup orelixir. A syrup or elixir, may comprise, for example, at least oneactive agent, a sweetening agent, a preservative, a flavoring agent, adye, a preservative, or combinations thereof.

In certain preferred embodiments an oral composition may comprise one ormore binders, excipients, disintegration agents, lubricants, flavoringagents, and combinations thereof In certain embodiments, a compositionmay comprise one or more of the following: a binder, such as, forexample, gum tragacanth, acacia, cornstarch, gelatin or combinationsthereof; an excipient, such as, for example, dicalcium phosphate,mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate or combinations thereof; a disintegratingagent, such as, for example, corn starch, potato starch, alginic acid orcombinations thereof; a lubricant, such as, for example, magnesiumstearate; a sweetening agent, such as, for example, sucrose, lactose,saccharin or combinations thereof; a flavoring agent, such as, forexample peppermint, oil of wintergreen, cherry flavoring, orangeflavoring, etc.; or combinations of the foregoing. When the dosage unitform is a capsule, it may contain, in addition to materials of the abovetype, carriers such as a liquid carrier. Various other materials may bepresent as coatings or to otherwise modify the physical form of thedosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both.

Additional formulations which are suitable for other modes ofadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum, vagina or urethra. After insertion, suppositoriessoften, melt or dissolve in the cavity fluids. In general, forsuppositories, traditional carriers may include, for example,polyalkylene glycols, triglycerides or combinations thereof In certainembodiments, suppositories may be formed from mixtures containing, forexample, the active ingredient in the range of about 0.5% to about 10%,and preferably about 1% to about 2%.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

P. KITS

Any of the compositions described herein may be comprised in a kit. In anon-limiting example, an immunoglobulin fusion protein, a nucleic acidcoding for an immunoglobulin fusion protein and/or additional agent, maybe comprised in a kit. The kits will thus comprise, in suitablecontainer means, a fusion protein, a nucleic acid coding for a fusionprotein and/or an additional agent of the present invention. Theinventors envisage other components that may be included in a kit. Theseinclude but are not limited to immunodetection agents such as peroxidaseand alkaline phosphatase linked monoclonal and polyclonal antibodies,immunoprecipitation reagents such as protein A- or protein G-linkedbeads, immune cell purification reagents such as magnetic beads, cloningreagents for the purpose of manipulating an expression vector, proteinexpression reagents including prokaryotic and eukaryotic cell lines forthe purpose of protein expression.

The kits may comprise a suitably aliquoted fusion protein and/oradditional agent compositions of the present invention, whether labeledor unlabeled, as may be used to prepare a standard curve for a detectionassay. The components of the kits may be packaged either in aqueousmedia or in lyophilized form. The container means of the kits willgenerally include at least one vial, test tube, flask, bottle, syringeor other container means, into which a component may be placed, andpreferably, suitably aliquoted. Where there is more than one componentin the kit, the kit also will generally contain a second, third or otheradditional container into which the additional components may beseparately placed. However, various combinations of components may becomprised in a vial. The kits of the present invention also willtypically include a means for containing the fusion protein, lipid,additional agent, and any other reagent containers in close confinementfor commercial sale. Such containers may include injection orblow-molded plastic containers into which the desired vials areretained.

Therapeutic kits of the present invention comprise an immunoglobulinfusion protein, polypeptide, peptide, inhibitor, gene, vector and/orother effectors. Such kits will generally contain, in suitable containermeans, a pharmaceutically acceptable formulation of an immunoglobulinfusion protein in a pharmaceutically acceptable formulation. The kit mayhave a single container means, and/or it may have distinct containermeans for each compound.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. The immunoglobulin fusionprotein composition may also be formulated into a syringeablecomposition, in which case, the container means may itself be a syringe,pipette, and/or other such like apparatus, from which the formulationmay be applied to an infected area of the body, injected into an animal,and/or even applied to and/or mixed with the other components of thekit.

However, the components of the kit may be provided as dried powder(s).When reagents and/or components are provided as a dry powder, the powdercan be reconstituted by the addition of a suitable solvent. It isenvisioned that the solvent may also be provided in another containermeans.

The container means will generally include at least one vial, test tube,flask, bottle, syringe and/or other container means, into which theimmunoglobulin fusion protein formulation is placed, preferably,suitably allocated. The kits may also comprise a second container meansfor containing a sterile, pharmaceutically acceptable buffer and/orother diluent.

The kits of the present invention will also typically include a meansfor containing the vials in close confinement for commercial sale, suchas, e.g., injection and/or blow-molded plastic containers into which thedesired vials are retained.

Irrespective of the number and/or type of containers, the kits of theinvention may also comprise, and/or be packaged with, an instrument forassisting with the injection/administration and/or placement of theultimate immunoglobulin fusion protein within the body of an animal.Such an instrument may be a syringe, pipette, forceps, and/or any suchmedically approved delivery vehicle.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Asused herein “another” may mean at least a second or more.

MG is aggregated IgG; IC is an immune complex; a HCH2 unit comprises thehinge and CH2 domain of an immunoglobulin; FcγR is Fc gamma receptor;SLE is systemic lupus erythematosus; MS is multiple sclerosis; CDCC iscomplement-dependent cellular cytotoxicity; ADCC is antibody-dependentcell-mediated cytotoxicity; CDC is complement-dependent cytotoxicity;EAE is experimental autoimmune encephalomyelitis; NK cells are naturalkiller cells; and PBMC are peripheral blood mononuclear cells.

Q. EXAMPLES

The following example is included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example I Framework Vector

The framework region of human IgG₁ comprised of H—CH2-CH3 was isolatedfrom total RNA from cell line ARH-77 and subcloned using RT-PCR, aprimer, FRM-5p-H3, that introduced a HindIII site immediately 5′ of thehinge region and a second primer, FRM-3p-Sal, which introduced a SalIsite immediately 3′ of the stop codon (Table 5). Clone pFRM-HS wascharacterized by DNA sequencing and used for further expressionconstruct assembly. Primers for this and subsequent steps involving IgG1cloning were designed using sequence data from the human IgG1 constantregion gene as a guide (accession #Z17370).

TABLE 5 Sequence of Primers used for PCR Amplification Name SequenceFRM-5P-H3 GgccgctaAAGCTTGAGCCCAAATCTTGTGACAA AACTC FRM-3P-SalGgccgctaGTCGACTCATTTACCCGGAGACAGGGAGAG Hinge1CccgtaGAATTCGAGCCCAAATCTTCTGACAAAACTCA CACATCCCCACCGTCCCCA CH2NH3GgccgcatAAGCTTggagccTCGCGATTTGGCTTTG GAGATGGTTTTCTC SMA-DELHGgccgcatCCCGGGGAGCCCAAATCTTCTGACAAAACT CH2H3GgccgcatAAGCTTTTTGGCTTTGGAGATGGTTTTCTC CD8-5PXhoGgccgctaCTCGAGATGGCCTTACCAGTGACCGCCTTG CD8-3P119EcoGgccgctaGAATTCCGTCGTGGTGGGCTTCGCTGGCAG The small letters indicate basesused as clamps or spacers. Bold face letters denote the location ofrestriction sites.

Example 2 Hinge Mutagenesis and CH2 Subcloning

The FcγR binding region of Fc, composed of the hinge and CH2 domains(HCH2), was isolated as a separate monomer unit. The hinge region withinthe HCH2 monomer unit was modified using PCR mutagenesis to change thethree cysteines that form inter-chain disulfide bridges between Fc unitsto serines. The FcγR binding domain was amplified using a 5′ primer,Hinge1 (Table 5), which introduced single nucleotide changes in each ofthe three hinge cysteine codons resulting in their alteration to serineresidues. The 5′ primer also introduced an EcoRI site immediately 5′ ofthe hinge region. The 3′ primer, CH2H3 (Table 5), directed theamplification of the CH2 domain and introduced an in-frame 3′ NruI siteseparated by a 6 nucleotide spacer from a HindIII site. The PCR productwas digested with EcoRI and HindIII and cloned into vector pBSKS+. Theconstruct, composed of 5′ EcoRI-HCH2-NruI -HindIII 3′, is termed “ENH”to denote the sequence of restriction sites and served as the startingunit for polymer construction. Clone pENH18 was characterized by DNAsequencing and used in subsequent cloning steps.

Two additional constructs, an extension unit designated pSNH, and acapping unit designated pSH3, were generated. These varied from pENH18only in their flanking restriction sites. pSNH has 5′SmaI-HCH2-NruI-HindIII 3′ and was amplified using pENH18 as template andprimers that introduced the flanking restriction sites (Table 5). Thesecond construct, pSH3, contains 5′ SmaI-HCH2-HindIII 3′ and wasamplified from pENH18 template using a 5′ primer, SMA-DELH, and a 3′primer, CH2H3 (Table 5), which introduced a single HindIII site thatflanks the 3′ end of the CH2 domain. Both the pSNH and pSH3 plasmidswere digested with SmaI and HindIII. The inserts were gel purified andstored for future use.

Example 3 Polymer Construction

Polymers composed of HCH2 units were built using the scheme presented inFIG. 1. The HCH2 polymers were constructed by the sequential addition ofa single starting unit (ENH), multiple extension units (SNH), and endedby addition of a single capping unit (SH3). Clone pENH18 was digestedwith NruI and HindIII resulting in a 5′ blunt end and a 3′ sticky end.Next a SNH insert, digested as described above, was ligated into thelinearized vector resulting in the in-frame insertion of a HCH2 repeatunit at the 3′ end of the pENH18 starting unit. The insertion alsoregenerated the original sequence of restriction sites(NruI—spacer-HindIII) which were used in the next round of extension.The extension process continued with NruI and HindIII digestion followedby ligation with the next SNH insert as described above. This cycle ofdigestion and insertion was repeated as needed to generate the linearpolymers. In the final round of polymer construction a ‘capping’ unit(SH3 insert) is ligated into the polymer instead of the SNH insert. Thisresulted in the loss of the internal cloning site. The result was thestepwise insertion of HCH2 units into the framework expression vector.Directionality of HCH2 insertion was maintained by the use ofnon-compatible flanking restriction sites. The junction between the HCH2units was composed of the fusion of the 5′ NruI half-site to the 3′ SmaIhalf-site, resulting in an in-frame Gly-Ser spacer between the proteindomains. The completed polymer constructs were liberated from the pBSKS+cloning vector at the EcoRI and HindIII sites and cloned intolike-digested pFRM-HS resulting in the in-frame joining of the HCH2polymers to the IgG₁ framework region.

Example 4 Cloning of the Extracellular Domain of Human CD8α

The secretion signal and first 119 residues of the extracellular domainof human CD8α were amplified using PFU polymerase (Stratagene), CD8αcDNA, and primers that introduced flanking 5′ XhoI and 3′ EcoRI sites(Table 5). Primers were designed using sequence data from the human CD8αcDNA as a guide (accession #M12824). The PCR product was digested withXhoI and EcoRI and cloned into like-digested pBlueBac4.5 baculovirustransfer vector (Invitrogen). The resulting construct, pCD8Bac, was usedas host for subsequent cloning steps.

The polymer-framework constructs were liberated from the pBSK S+ cloningvector by digestion with EcoRI and SalI and united with the CD8αsequences by their ligation into like-digested pCD8Bac. The CD8-HCH2polymer constructs were liberated from pCD8Bac at the BamHI and SalIsites, gel purified and ligated into the same sites in the pIE1-4 insectcell expression vector (Novagen). The pIE1-4 vector had been modifiedpreviously to accommodate the restriction sites that flank the inserts.The resulting expression constructs have the CD8α secretion signal andextracellular domain on their 5′ termini fused to the HCH2 polymer unitsin the middle and the framework domains on their 3′ termini (FIG. 2).

Example 5 Establishment of Stable Expressing Polyclonal Cell Lines

Plasmid DNA destined for transfection was purified using Qaigen plasmidDNA isolation columns (Qaigen). The pelleted DNA was washed repeatedlywith 70% ethanol, air dried and resuspended in sterile TE buffer. Sf9insect cells (ATCC) were propagated in ExCell 420 medium (JRHBiosciences) containing 100 u/mL penicillin and 100 μg/mL streptomycin(Gibco). One day prior to transfection, 2×10⁶ cells were plated onto 60mm culture dishes in ExCell 420 medium supplemented with 10% Sf9conditioned medium.

Sf9 cells were washed once and the medium replaced with 2 mLantibiotic-free ExCell 420. Transfection was mediated by the cationiclipid Cellfectin (Gibco). 5 μg of expression construct along with 1 μgof pIE-Neo or 6 μg of pBSKS as a negative control were added to 290 μLof antibiotic-free ExCell 420. In a separate tube 280 μL ofantibiotic-free medium was mixed with 20 μL of Cellfectin reagent. Thecontents of the tubes were combined and the DNA-lipid complexes wereallowed to form over a period of 30 min after which time they were addeddrop-wise to each 60 mm culture dish. Cells were incubated for 8 hoursafter which time 2 mL of medium was added. Incubation with the DNA-lipidcomplexes continued overnight. On the day after transfection the mediumwas removed and replaced with antibiotic containing medium supplementedwith 10% Sf9 conditioned medium. On the second day post-transfection thecells were split into T25 flasks with selection medium composed ofExCell 420 supplemented with 10% conditioned Sf9 medium, 400 μg/mL ofgeneticin (G418)(Gibco), and antibiotics. Flasks were monitored for celldeath, the pBSKS control cells died within 10 days pIE-Neo containingtransfectants showed robust growth. The cultures were expanded into T75flasks and used as seed stocks for protein expression.

Example 6 Protein Expression and Purification

To express larger amounts of protein, 250 mL cultures were initiated inspinner and/or shaker flasks. Cultures were grown in ExCell 420 with 100μg/mL G418, 01% pluronic F-68, and antibiotics. Culture supernatantswere centrifuged to remove cellular debris. PMSF (Sigma) and Pepstatin A(Sigma) were added to a final concentration of 1 mM and 1 μMrespectively.

Conditioned medium was clarified by passage through a 0.45 μm filter andapplied to 1 mL protein G-sepharose (Pharmacia) columns at a rate of 1mL/min. Columns were washed with 100 mL PBS, pH 7.0 and proteins elutedwith 3 mL of elution buffer (20 mM glycine, 150 mM NaCl, pH 3.0). Eluatewas immediately brought to neutral pH by the addition of 100 μL of 1 MTris, pH 9.0. Recombinant proteins were equilibrated in RPMI medium andconcentrated using centrifugal concentrators with a MW cutoff of 30 kD(Amicon/Millipore). Protein concentrations were determined using theBradford method (Biorad) with human IgG as the standard.

Example 7 Expression of HCH2 Polymers Fused to an Amino Terminal HumanCD8α Domain

To demonstrate the utility of the vectors, the extracellular domain ofhuman CD8α was expressed as a HCH2 polymer fusion protein. The secretionsignal and first 119 residues of human CD8α were cloned into the aminotermini of the HCH2 polymers. The expression constructs were insertedinto the pIE1-4 vector (Novagen), which places the fusion constructsunder the control of the baculovirus ie1 gene promoter which isconstitutively active in Sf9 cells (Jarvis, et al., 1996). The use oftransfected insect cells can result in improved expression ofglycosylated secretory proteins due to more efficient processing andsecretion than is achieved in virus infected cells (McCarroll, et al.,1997; Pfeifer, et al., 1998).

Four constructs were chosen for expression analysis, termed CD8R0through CD8R4, which contain between 0 and 4 HCH2 units in the polymerin addition to the HCH2 unit within the framework (Table 6 and FIG. 2).As result of the covalent dimerization of the framework domains, themature proteins contain between 2 and 10 HCH2 units in the CD8R0 throughCD8R4 proteins respectively (Table 6). The fusion proteins were secretedin useful amounts from polyclonal cell lines established in Sf9 cellsfollowing transfection and selection with G418. The recombinant proteinswere isolated in a single step from conditioned culture medium bypassage over a protein G-sepharose column. The expressed polymers arestable, secreted, soluble and are readily concentrated to useful levels.The proteins are glycosylated, as documented by the difference inpredicted and observed molecular weights. Yields correlate inverselywith protein size and fall in the range of 0.8 to 2.0 μg/mL ofconditioned medium,

TABLE 6 Number of HCH2 units, potential N-linked glycosylation sites,predicted molecular weights, and contribution of N-linked glycosylationto apparent molecular weight of CD8-HCH2 polymers fused to the IgG1-Fcframework. Number Number Number of of of HCH2 HCH2 HCH2 units unitsinN-Linked Apparent units in single mature glycosylation Predicted MW (KD)Construct inserted chain polypeptide sites MW (KD) Control PNGase +CD8R0 0 1  2 2 39.5  44.2  43.3 CD8R2 2 3  6 4 67.8  85.7  82.1 CD8R3 34  8 5 82.0 104.8  97.5 CD8R4 4 5 10 6 96.2 125.7 116.9

Example 8 Structural Integrity

To examine the structural integrity and antigenic content, therecombinant proteins were resolved on SDS-PAGE gels and analyzed byWestern blot. Proteins were electrophoresed on 7% SDS-PAGE gels (Laemmliet al., 1970) and transferred to nitrocellulose membranes (MSI).Membranes were blocked overnight in 5% non-fat milk in Tris-bufferedsaline, pH 7.4 (TBS). For analysis of Fc domains, a total of 50 ng ofrecombinant protein or 0.5 μg of control proteins (human IgG and BSA)were loaded onto the gels. The membrane was incubated for two hours withhorse radish peroxidase (HRP)-labeled goat anti-human Fc polyclonalantibody (Caltag) used at 1:10000 dilution in a binding bufferconsisting of 0.1% non-fat milk and 0.1% normal goat serum in TBS. Theblot was washed with TBS-tween and detection performed using theECL-plus chemoluminescent reagent following manufacturers instructions(Amersham).

For CD8α Western blot analysis, recombinant proteins were loaded ontogels at 200 ng and controls were loaded at 0.5 ug per well. The membranewas incubated for two hours with mouse anti-human-CD8α monoclonalantibody (clone HIT8a, Pharmingen) used at 1:800 dilution in a bindingbuffer consisting of 0.2% non-fat milk and 0.1% normal human serum inTBS. Blots were washed as above and incubated with HRP conjugated rabbitanti-mouse IgG (DAKO) used at 1:1000 dilution in a binding bufferconsisting of 0.1% non-fat milk, 0.1% normal human serum, 0.1% normalgoat serum, and 0.03% Tween in Tris-buffered saline. Blots were washedand detected as above. For direct visualization of proteins, gels werestained with Coomassie brilliant blue.

Results: As shown in FIG. 3A the HCH2 polymers are expressed, stable,and secreted. The observed molecular weight is larger than predicted forthe peptide backbone alone (FIG. 2), which indicates that the proteinsare glycosylated (see also Table 6). A Western blot probed withantibodies directed against human Fc reveals binding to the HCH2polymers in a fashion similar to the IgG control (FIG. 3B). A similarblot probed with antibodies directed against human CD8α shows bindingonly to recombinant protein (FIG. 3C). Taken together, these resultsdemonstrate that the HCH2 polymers contain both the CD8α and Fcantigenic determinants, indicating that the proteins, are correctlyexpressed and secreted. Correlation of the amount of purified protein tooriginal volume of conditioned medium gives yields that range from 2μg/mL (CD8R0) to 0.8 μg/L (CD8R4) of culture medium. Yields correlatednegatively with protein size and/or the number of HCH2 repeat units. Noevidence for accumulation of misfolded or aggregated recombinantproteins was found in the Sf9 cell pellets implying some other basis forthe bias against larger proteins.

Example 9 Dimerization of HCH2 Polypeptides using Disulfide Linkages

One anticipated difficulty was the potential for mutated HCH2 units tocompete with native hinge regions during oligomerization. This couldpotentially result in a significant amount of monomer production. Theproteins were analyzed on SDS-PAGE gels under reducing and non-reducingconditions to determine if the HCH2 polymers form antibody-like covalentoligomers. In all cases the recombinant proteins formed dimers thatcould be reduced by 2-mercaptoethanol indicating that IgG-likeoligomerization had occurred However, both CD8R0 and CD8R4 produceddetectable levels of monomers. Whether this resulted from post-secretiondisulfide-bond reduction or a failure to oligomerize remains unresolved.The presence of monomers in the CD8R0 preparations argues against acompetitive mechanism however.

Example 10 Cloning and Expression of Human Serum Albumin (HSA) Domain IFused to HCH2 Polymers

Previously, HCH2 polymers had been expressed as fusions with theextracellular domain of human CD8α. In order to discern which effectsare attributable to the HCH2 polymers and which to the amino-terminalfusion partner, fusion proteins were constructed with the domain I ofHSA fused to the HCH2 polymers. The biological activities of theCD8α-HCH2 polymers could then be compared to those of the HSA-HCH2polymers. These experiment also serve to demonstrate the general utilityof the expression system. The secretion signal and first 197 residues ofdomain I of HSA were amplified using RT-PCR, total RNA derived from cellline HEP G2 (ATCC HB-8065), and primers that introduced flanking 5′ XhoIand 3′ EcoRI sites. Primers were designed using sequence data from theHSA cDNA as a guide (accession # V00494). The PCR product was digestedwith XhoI and EcoRI and cloned into like-digested pFRM-HCH2 vectors. ThepFRM-HCH2 vectors direct expression of N-terminal protein domains fusedin-frame to HCH2 polymers with varying numbers of HCH2 repeats. Thevector backbone is derived from the pIE1-4 insect cell expression vector(Novagen) which places fusion protein constructs under the control ofthe baculovirus ie1 gene promoter.

The HSA-HCH2 fusion constructs were stably transfected into SF9 cells aswas done previously for the CD8α-HCH2 constructs. Similarly, fourconstructs were chosen for expression analysis, termed HSAR0 throughHSAR4, which contain between 0 and 4 HCH2 units in addition to the HCH2unit within the framework (FIG. 2). As a result of the covalentdimerization of the framework domains, the mature proteins containbetween 2 and 10 HCH2 units in the HSAR0 through HSAR4 proteinsrespectively. Recombinant protein was isolated from conditioned mediumas described in Example 6 above. The proteins were resolved on 7%SDS-Page gels and stained to reveal protein.

Results: HSA-HCH2 fusion proteins are expressed, secreted, and stable.The proteins can be isolated from conditioned medium and concentrated touseful levels. Yields are comparable to those observed for CD8αconstructs. These results demonstrate the general utility of the HCH2expression vectors with two different fusion partners. Also the resultsdemonstrate the stability of the expressed HCH2 fusion proteins withdifferent fusion partners_(—) Experiments described below evaluate andcompare the relative biological activities of the recombinant proteins.

Example 11 PBMC Purification, Proliferative Assays, and CytokineAnalysis

Peripheral blood mononuclear cells (PBMC) of six healthy donors wereisolated from heparinized blood on a Ficoll-Paque gradient (PharmaciaBiotech Inc) and suspended in AIM V defined serum free medium (GibcoBRL). Recombinant protein stocks were initially prepared in RPMI 1640(concentration ≧1 mg/ml). Recombinant protein stocks were diluted in AIMV medium (Fisher Scientific) to achieve the desired final concentrationsas indicated in the drawings. To prepare IgG aggregates, 4 mg of humanIgG (Organen Teknika Corp.) was dissolved in 2 ml of saline andincubated at 57° C. for one hour. The heat aggregated IgG (MG) obtainedwas diluted in AIM V medium to the desired final concentration asindicated in the drawings. rIL-2 (Pharmingen) was added at a finalconcentration of 1 ng/ml. To stimulate PBMC with the anti-CD16 mAb 3G8(Caltag Laboratories), 50 μl of mAb 3G8 (10 μg/ml or dilutions thereof)in bicarbonate buffer (pH 8.4) was overlaid in each well of a 96 wellflat bottom tissue culture plate (Corning Costar) for three hours at RT.The wells were then thoroughly washed with saline, aspirated, and usedimmediately thereafter for cell culture. PBMC were plated at a finalconcentration of 2×10⁶ cells/ml in 96 well flat bottom plates (0.200ml/well final volume) or 48 well flat bottom plates (1 ml/well finalvolume). For cytokine induction, cells were incubated for 48 hours in ahumidified incubator at 37° C. in 5% atmospheric CO₂ and the supernatantharvested and centrifuged at 2100 rpm for 10 minutes to pellet cells andcell debris. The cell free supernatant was frozen at −80° C. untilassayed for cytokine content. For proliferative assays, cells wereincubated for 72 hours in a humidified incubator at 37° C. in 5%atmospheric CO₂. During the last 5 hours of culture, wells were pulsedwith 1 μCi of [methyl-³H] thymidine (Amersham Corp). Cells wereharvested using a PhD cell harvester (Cambridge Technologies).Radioactivity was determined using a Beckman Scintillation Counter LS5000TD (Beckman Instruments).

Example 12 IFN-γ and TNF-α ELISA

To detect IFN-γ and TNF-α a sandwich ELISA was used. To detect IFN-γ,the mouse anti-human antibody clone NIB42 was used as the captureantibody and the antibody clone 4S.B3 was used as the detecting antibody(both from Pharmingen Corporation). To detect TNF-α, the mouseanti-human antibody clone MAb1 was used as the capture antibody and theantibody clone MAb11 was used as the detecting antibody (both fromPharmingen Corporation). ELISA, plates (Costar Corporation, Cambridge,Mass.) were coated with 0.1 ml/well of capture antibody at 0.75 μg/ml incarbonate buffer (0.5 M, pH 8.5). Plates were incubated overnight atroom temperature (RT). 0.100 ml of 2% crystallized BSA in Dulbecco'sphosphate buffered saline (DPBS) was added to each well for anadditional 3 to 6 hours at room temperature. Plates were washedextensively with DPBS and recombinant IFN-γ or TNF-α (used as standards,Pharmingen Corp.) or cell supernatants were added. Recombinant IFN-γ orTNF-α was serially diluted 1:3 from 10 ng/ml to 0.15 ng/ml. Cellsupernatants were assayed at 50% and 10% dilutions. Plates wereincubated overnight at 4° C., wells were washed, and overlaid with 0.200ml of biotinlyated detecting antibody at 0.75 μg/ml in 0.2% BSA in DPBSfor 2 hours at room temperature. Wells were washed and overlaid with0.200 ml of goat polyclonal affinity purified IgG reactive to biotin(1:400 in 1% DPBS, Zymed Laboratories, South San Francisco, Calif.) for1 hour at RT. Wells were washed and 0.200 ml of ortho-phenylenediamine(4 mg/ml) in citrate buffer (0.1 M, pH 4.5) was added to each well.Plates were read on a Thermomax Microplate reader (Molecular DevicesCorp. Menlo Park, Calif.).

Example 13 Assessment of HCH2 Polymer—FcγRIII Interactions

To assess potential HCH2 polymer—FcγRIII interactions, HCH2-polymerswere assayed for their ability to activate NK cells within PBMCisolates. NK cells express both the low affinity IL-2 receptor, andFcγRIII (CD16) (Nagler et al., 1990). When primed with high levels ofIL-2 (1 ng/mL), NK cells mount a proliferative response to CD16ligation. This triggered response was used as a test of the fitness ofIC, MG and recombinant molecules to engage FcγR. Two different sets ofHCH2 polymer constructs were tested for their ability to activate PBMC,one expressing the extracellular domain of CD8α (referred to as CD8R0,CD8R2, CD8R3, and CD8R4) and the other expressing the domain one of HSA(referred to as HSAR0, HSAR2, HSAR3, and HSAR4).

-   RESULTS: Immobilized anti-CD16 mAb 3G8, in the presence of 1L-2,    triggers NK cell proliferation and cytokine release through    activation of the FcγIII receptor. PBMC isolates were incubated with    medium alone, IL-2 (1 ng/ml) alone, immobilized anti-CD16 mAb alone,    or with IL2 plus anti-CD16 mAb, and proliferative responses measured    3 days later. Neither IL-2 nor anti-CD 16 mAb alone induce    significant proliferative responses from PBMC. Proliferative    responses in the presence of medium alone were 787±447, in the    presence of IL-2 alone were 1957±1117, and in the presence of    immobilized anti-CD16 mAb alone were 592±102. However there was a    marked increase in proliferative response in the presence of both    IL-2 and anti-CD16 mAb. Proliferative responses in the presence of    both IL-2 and immobilized anti-CD 16 mAb were 15499±2962. Data    represent the average from four individuals±SEM.

Similarly, HCH2 polymer constructs, expressing the extracellular domainof CD8α were also tested for their ability to induce proliferativeresponses in PBMC. As shown in Table 7, the CD8α expressing constructs,CD8R0, CD8R2, CD8R3, and CD8R4, all induce proliferative responses inPBMC in the presence of IL-2. Thus, the ability to induce PBMCproliferation correlates with the number of HCH2 units indicating thatthe constructs mimic MG function. As CD8R4 was the most effectiveconstruct in the assay, as documented in Table 7, it was compared to AIGdirectly. CD8R4 was as effective at 5 μg/mL as AIG at 125 μg/mL andproliferation was of the same magnitude as that observed using anti-CD16mAb. Proliferative responses in the presence of 5 μg/mL of CD8R4 andIL-2 were 21694±4636 (18 fold induction over IL-2 alone) and in thepresence of 125 μg/mL of AIG and IL-2 were 17388±1342 (16 fold inductionover IL-2). HCH2 polymers containing the first domain of HSA were alsotested for their ability to induce proliferative responses in PBMC. Asshown in FIG. 4., the HCH2 polymer protein HSAR4 was as effective inactivating PBMC to proliferate as was immobilized anti-CD16 Ab 3G8 andmany times more effective than aggregated IgG.

TABLE 7 Proliferative responses from costimulation of PBMC withrecombinant HCH2 polymers and IL-2 Construct Induction (CPM)* IL-2 Alone 2011 ± 714 CD8R0 13036 ± 3339 CD8R2 17696 ± 3876 CD8R3 19293 ± 3412CD8R4 21010 ± 3425 Significance R0 vs R2 < 0.019 (Student's paired ttest) R0 vs R3 < 0.025 R0 vs R4 < 0.0097 R2 vs R3 < 0.18 R2 vs R4 <0.036 R3 vs R4 < 0.15 *Mean ± SEM

Example 14 PBMC Activation by HCH2 Polymer Proteins With Varying HCH2Repeat Units

PBMC activation by HCH2 polymer proteins correlates with the number ofHCH2 region repeats indicating a high level of sensitivity by Fcγreceptors to HCH2 number in the HCH2 polymer proteins (FIG. 5). PBMCwere exposed to varying concentrations of different HCH2 polymerproteins containing varying numbers of HCH2 domains in the presence ofIL-2 (FIG. 5), RESULTS: A dose dependent response is observed for eachHCH2 polymer protein. PBMC respond better to HCH2 polymer proteinscontaining greater numbers of repeating HCH2 units. Thus, HCH2 polymerproteins can be utilized to discern subtle differences in receptorreactivity to Ig. As indicated in the legend of FIG. 5, proliferativeresponses to the HCH2 polymer proteins in the absence of IL-2approximated those found in medium alone. Thus, the ability of the HCH2polymer proteins to activate PBMC to proliferate is dependent uponco-stimulation with IL-2 as observed with immobilized anti-CD16 Ab. Datashown represent the means obtained using PBMC from four differentdonors.

Example 15 HCH2 Polymer Proteins that Express Different Protein DomainsActivate PBMC in a Similar Manner

To directly compare the biological function of HCH2 polymer proteinsconstructed with repeating HCH2 units along with domains from differentproteins, the ability of HCH2 polymer proteins expressing domain one ofHSA (HSAR0 and HSAR4) to activate PBMC was compared to that of HCH2polymer proteins expressing the extracellular domain of CD8 alpha (CD8R0and CD8R4) Table 8. HSAR0, HSAR4, CD8R0, and CD8R4 were used at aconcentration of 5 μg/ml along with IL-2 (1 ng/ml) to activate PBMC asdescribed in Example 8. RESULTS: As shown in Table 8, HSAR0, HSAR4,CD8R0, and CD8R4 all activate PBMC to proliferate in the presence ofIL-2. An increase in the proliferative response is noted with constructscontaining higher numbers of HCH2 repeat units irrespective of thecoexpressed protein domain. Thus HCH2 polymer proteins may beconstructed with varying numbers of HCH2 repeat units that coexpressdomains from different types of proteins and still retain the biologicalactivating properties of the HCH2 repeat units.

TABLE 8 Proliferative responses from costimulation of PBMC withrecombinant HCH2 polymers and IL-2 Culture Condition cpm ± SEM Medium653 ± 179 IL-2 7132 ± 2423 HSAR0 + IL-2 5219 ± 1125 HSAR4 + IL-2 21837 ±4868  CD8R0 + IL-2 15991 ± 3320  CD8R4 + IL-2 25684 ± 4636 

Example 16 Cytokine Secretion Following Stimulation with HCH2 PolymerProteins

HCH2 polymer proteins activate PBMC to secrete cytokines in a mannersimilar to the natural IgG₁ ligand and anti-CD16 mAb (Table 9). PBMCwere activated with IL-2 alone or IL-2 plus immobilized anti-CD16antibody 3G8 (10 μg/ml), aggregated IgG (40 μg/ml), or HSAR4 (40 μg/ml)(Table 9). Both IFNγ and TNFα production by PBMC increase in thepresence of anti-CD16 Ab, AIG, and HSAR4 compared to 1-2 alone.

TABLE 9 Cytokine secretion from PBMC stimulated with IL-2 alone, or withimmobilized anti-CD16 mAb, aggregated IgG, or HSAR4 TNF-α ± SEM IFN-γ ±SEM (ng/ml) (ng/ml) Medium 0.01 ± .01  0 ± 0 IL-2 0.80 ± 0.4  2.7 ± 2.1Immobilized anti-CD16 mAb + IL-2 3.32 ± 1.49 9.85 ± 3.38 AggregatedIgG + IL-2 2.46 ± 0.76 13.01 ± 4.30  HSAR4 + IL-2 4.94 ± 1.03 33.58 ±2.53 HCH2 polymer proteins induce IFNγ and TNFα secretion from PBMC in a dosedependent manner (Table 10).

TABLE 10 Cytokine secretion following stimulation with HCH2 polymerproteins. HSAR0 (μg/ HSAR4 (μg/ ml) + IL-2 ml) + IL-2 IL-2 10 5 1 10 5 1alone medium IFN-γ 1.6  .9  .3 2.6 2.4 1.7 .18 .15 TNF-α  .34 .17 .2 1.91.5 1.2 .2  .18PBMC were exposed to varying concentrations of two HCH2 polymerconstructs, one, HSAR0, has no inserted HCH2 domains though as a resultof the dimerization of the Fc framework region there are two HCH2domains in the mature polypeptide, the other, HSAR4, has a total of tenHCH2 domains in the mature polypeptide (see Table 6). Both IFNγ and TNFαsecretion were induced in PBMC stimulated with both constructs. At thesehigh concentrations it is likely that some of the HSAR0 constructbecomes immobilized on the well surface and effectively stimulateFcγRIIIa. At lower concentrations of protein, only HCH2 polymer HSAR4induces cytokine secretion showing the effect of the additional HCH2domains on receptor activation.

Example 17 HCH2 Polymers can be Expressed to Minimize Interaction withComplement Factor C1q

For certain therapeutic applications, the binding of complement to theHCH2 polymers could pose an unwanted and potentially deleterious sideeffect. In addition, complement binding to the HCH2 polymers couldconfound results in certain studies. Insect cells are known to expressproteins that have altered carbohydrate moieties. These alterations mayweaken binding of complement factor C1q to these proteins. For thisreason the binding of C1 q to HCH2 polymers expressed in insect cellline SF9 was investigated. An assay examining the binding of C1q tohuman IgG or to HCH2 polymers expressed in insect cells was undertaken.Various concentrations of human C1q were allowed to bind to either humanIgG or to the HCH2 polymers HSAR0 and HSAR4 previously immobilized ontowells of a 96 well ELISA plate. The extent of C1q binding was detectedusing a goat anti-human C1q polyclonal antibody. The results, showngraphically in FIG. 6, demonstrate that HCH2 polymers isolated from aninsect cell expression system engage C1q more weakly than native IgG.

Example 18 EAE Induction

EAE was induced in SJL/J mice, six to seven weeks old. Each mousereceived a total of 0.1 ml of adjuvant distributed over three sites onthe back. Injections were delivered intradermally into shaved regions ofthe skin above the flanks and between the shoulder blades. To prepareadjuvant for immunization, myelin proteolipid protein peptide 139-151(Peptides International, Louisville, Ky.) was dissolved in PBS at 1.5μg/ml and emulsified with an equal volume of Complete Freund's adjuvant(Difco Laboratories, Detroit, Mich.). 200 ng of pertussis toxin (ListBiological Labs Inc., Campbell, Calif.) in 0.1 ml of saline was injectedinto the tail vein of each mouse 1 day and 3 days after immunization. Todetermine the effect of the constructs on disease severity, mice wereinjected intraperitoneally with saline alone (150 μl total volume) orwith saline containing HSAR0 (50 μg HSAR0/150 μl of saline) or withsaline containing HSAR4 (50 μg HSAR4/150 μl of saline). Mice wereinjected with the constructs or saline control, 3 days beforeimmunization, 1 day after immunization and 3 days after immunization.Clinical disease was graded on a scale of 0 to 5 of increasing severity;0, no abnormality; 1, a flaccid tail; 2, a flaccid tail with mild hindlimb weakness; 2.5, moderate hind leg weakness but not completeparalysis; 3, total paralysis of hind legs, 4, hind leg paralysis withforelimb weakness or paralysis; 5, moribund. Mice that became moribundwere euthanized.

EAE in the SJL/J mouse strain is characterized by an early acute diseasefrom which the mice recover partially or fully. The disease thenrelapses and becomes a chronic relapsing illness from which the miceseldom recover. RESULTS: As shown in FIG. 7, mice injected with HSAR4displayed a less severe acute disease compared to mice injected withsaline alone or with HSAR0. Mice injected with HSAR0 had an acutedisease of intermediate severity compared to mice injected with HSAR4 orsaline alone. All mice injected with HSAR4 or HSAR0 recovered from theacute illness while the majority of those treated with saline alonenever recovered fully from the acute phase of the disease. Mice injectedwith HSAR4 displayed fewer relapses than mice treated with HSAR0 orsaline alone. Data shown are the average clinical disease scores. N=8mice for saline, n=8 for HSAR0 and n=6 for HSAR4. One mouse treated withHSAR0 became moribund on day 14 and was euthanized. A score of 5 wasentered into the data for day 14 and thereafter entries were not madefor this animal.

Mice treated with HSAR4 had significantly less severe disease during theacute phase and during the relapsing phase of the disease than didsaline controls and this persisted for longer periods of time and to agreater extent than that observed for HSAR0 treated animals (p<0.05 vs.saline for each day of observation from the onset of disease on day 8 today 13, and from days 15 to day 41, non-inclusive of day 26; unpairedstudent's t test). HSAR0 treated mice had significantly less severedisease compared to saline treated controls during both the acute phaseof disease and during relapses (p<0.05 vs. saline for each day ofobservation from days 9 to 12, and from 16 to 19.5; unpaired student's ttest). Significantly less disease activity was also observed at latertime points in both the HSAR0 and HSAR4 treated groups compared tosaline controls.

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The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. A method of treating a mammal comprising:administering a polypeptide to the mammal, where the mammal has aninflammatory disease or an autoimmune disease, and the polypeptideconsists of a dimer of a first amino acid chain and a second amino acidchain, which can be the same or different, and where each amino acidchain comprises: a first region comprising a protein or portion thereof;a second region comprising two to six copies of at least a portion of anHCH2 region of a first mammalian IgG, where the HCH2 region comprises ahinge of the first mammalian IgG and a CH2 of the first mammalian IgG;and a third region comprising a hinge of a second mammalian IgG, a CH2of the second mammalian IgG, and a CH3 of the second mammalian IgG;wherein (1) one or more cysteines of the hinge of the HCH2 region of oneor both amino acid chains is mutated to an amino acid selected from thegroup consisting of serine, alanine, glycine, and threonine, (2) thefirst mammalian IgG and the second mammalian IgG are from the samemammal, and (3) the polypeptide targets to cells expressing a lowaffinity FcγR or binds to a low affinity FcγR.
 2. The method of claim 1,wherein the mammal has the autoimmune disease.
 3. The method of claim 1,wherein the administering is selected from the group consisting oforally, intranasally, and via injection.
 4. The method of claim 1,wherein the administering is selected from the group consisting of viaintravenous injection, via intraperitoneal injection, via intramuscularinjection, and via subcutaneous injection.
 5. The method of claim 1,wherein the administering comprises administering about 0.75 mg ofpolypeptide/kg body weight.
 6. The method of claim 1, wherein the mammalis a human or a rodent.
 7. The method of claim 1, wherein the mammal hasthe inflammatory disease.
 8. The method of claim 1, wherein the methodcomprises treating the autoimmune disease.
 9. The method of claim 1,wherein the method comprises treating a disease selected from the groupconsisting of a disease of the platelets, multiple sclerosis, rheumatoidarthritis, lupus erythematosis, myasthenia gravis, and immunethrombocytopenic purpura.
 10. The method of claim 1, wherein the methodcomprises altering immunity in a mammal.
 11. The method of claim 1,wherein the method comprises treating the inflammatory disease.
 12. Themethod of claim 1, wherein the treating results in complement-dependentcytotoxicity.
 13. The method of claim 1, wherein the first regioncomprises an antigen binding portion of an antibody.
 14. The method ofclaim 1, wherein the first region binds to one or more selected from thegroup consisting of a tumor, a virus, a fungus, a rickettsia, amycoplasma, a bacterium, a protozoal parasite, and a metazoal parasite.15. The method of claim 1, wherein the first region is conjugated to aprotein or non-protein molecule derived from one or more selected fromthe group consisting of a tumor, a virus, a fungus, a rickettsia, amycoplasma, a bacterium, a protozoal parasite, and a metazoal parasite.16. The method of claim 1, wherein all of the cysteines of the hinge ofthe HCH2 region are mutated to an amino acid selected from the groupconsisting of serine, alanine, glycine, and threonine.
 17. The method ofclaim 1, wherein all of the cysteines of the hinge of the HCH2 regionare mutated to serine.
 18. The method of claim 1, wherein the firstregion is linked to the second region by a protein domain and/or alinker group.
 19. The method of claim 1, wherein the second region islinked to the third region by a protein domain and/or a linker group.20. The method of claim 1, wherein the second mammalian IgG is amammalian IgG selected from the group consisting of human IgG1, humanIgG2, and human IgG3.
 21. The method of claim 1, wherein the polypeptidetargets to cells expressing one or more low affinity FcγR selected fromthe group consisting of FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, andFcγRIIIb.
 22. The method of claim 1, wherein the polypeptide binds toone or more low affinity FcγR selected from the group consisting ofFcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIb.
 23. The method ofclaim 1, wherein the polypeptide is expressed in insect cell line SF9.24. The method of claim 1, wherein the HCH2 region of the firstmammalian IgG comprises at least SEQ ID NO: 10 and the one or morecysteine mutations occurs in at least one of the hinge cysteines in SEQID NO:
 10. 25. The method of claim 1, wherein the HCH2 region of thefirst mammalian IgG comprises at least SEQ ID NO: 10 and the one or morecysteine mutations occurs in the three hinge cysteines in SEQ ID NO: 10.26. The method of claim 1, wherein the polypeptide induces proliferationof PBMCs with greater potency than aggregated IgG.
 27. The method ofclaim 1, wherein the binding to the low affinity FcγR occurs withoutaggregation of the polypeptide or without formation of immune complexes.28. The method of claim 1, wherein the first region of the first aminoacid chain is different from the first region of the second amino acidchain.
 29. The method of claim 1, wherein the protein or portion thereofis selected from the group consisting of HSA, domain I of HSA (SEQ IDNO: 11), domain II of HSA (SEQ ID NO: 12), domain III of HSA (SEQ ID NO:13), CD8α, CD8β, and CD8.
 30. The method of claim 1, wherein the proteinor portion thereof is domain I of HSA (SEQ ID NO: 11).
 31. The method ofclaim 1, wherein the protein or portion thereof comprises at least aportion of a sequence selected from the group consisting of a CD8a, anHSA (SEQ ID NO: 14), and a transporter protein.
 32. The method of claim1, wherein the protein or portion thereof comprises at least a portionof a sequence from HSA (SEQ ID NO: 14).
 33. The method of claim 1,wherein the protein or portion thereof comprises at least a portion of asequence from myelin basic protein.
 34. The method of claim 1, whereinthe protein or portion thereof comprises at least a portion of asequence from proteolipid protein.
 35. The method of claim 1, whereinthe protein or portion thereof comprises at least a portion of a proteinselected from the group consisting of a toxin antigen sequence, a viralantigen sequence, and a tumor antigen sequence.
 36. The method of claim1, wherein the first mammalian IgG is the same as the second mammalianIgG.
 37. The method of claim 1, wherein the treating results in antibodydependent cellular cytotoxicity.
 38. The method of claim 1, wherein theprotein or portion thereof comprises a soluble protein domain, atransporter protein domain, a ligand binding domain, a cell specificreceptor, a cell specific binding site, a cell surface receptor domain,or a cell surface marker binding site.
 39. The method of claim 1,wherein one or more cysteines of the hinge of the HCH2 region aremutated to an amino acid selected from the group consisting of serine,alanine, and glycine.
 40. The method of claim 1, wherein all of thecysteines of the hinge of the HCH2 region are mutated to an amino acidselected from the group consisting of serine, alanine, and glycine. 41.The method of claim 1, wherein the HCH2 region of the first mammalianIgG is an HCH2 region of a mammalian IgG selected from the groupconsisting of human IgG1, human IgG2, and human IgG3.
 42. The method ofclaim 1, wherein the HCH2 region of the first mammalian IgG is from ahuman IgG1 and comprises at least SEQ ID NO:
 9. 43. The method of claim1, wherein the HCH2 region of the first mammalian IgG is from a humanIgG1 and comprises at least SEQ ID NO:
 10. 44. The method of claim 1,wherein the polypeptide comprises an Fc region.
 45. The method of claim1, wherein the first amino acid chain and the second amino acid chainare the same.
 46. The method of claim 1, wherein the protein or portionthereof comprises a soluble protein domain, a transporter proteindomain, a ligand binding domain, a cell specific receptor, a cellspecific binding site, a cell surface receptor domain, and/or a cellsurface marker binding site.
 47. The method of claim 1, wherein thefirst region comprises at least a portion of a sequence selected fromthe group consisting of an Fab region of a human antibody, an Fab regionof a humanized antibody, an antibody-like molecule, a CD8α, an HSA, anda transporter protein.
 48. The method of claim 1, wherein thepolypeptide is adapted to mimic functional interactions of aggregatedIgG and immune complexes with FcγR.
 49. The method of claim 1, whereinthe first region comprises at least a portion of a sequence selectedfrom the group consisting of a binding site for an antigen, a bindingsite of a cellular receptor, a binding site of a receptor ligand, and abinding site of an adhesion molecule.
 50. The method of claim 1, whereinthe first region comprises at least a portion of an Fab sequence able tobind to a tumor expressed antigen.
 51. The method of claim 1, whereinthe first mammalian IgG and the second mammalian IgG are selected fromthe group consisting of a human IgG, rodent IgG, cow IgG, goat IgG,sheep IgG, horse IgG, dog IgG, cat IgG, and pig IgG.
 52. The method ofclaim 1, wherein the first mammalian IgG and the second mammalian IgGare murine IgG.
 53. The method of claim 1, wherein the first mammalianIgG and the second mammalian IgG are human IgG.
 54. The method of claim1, wherein the polypeptide comprises an amino acid sequence selectedfrom the group consisting of IgG1, IgG2, and IgG3.
 55. The method ofclaim 1, wherein the polypeptide comprises an amino acid sequencecomprising a sequence of at least two selected from the group consistingof IgG1, IgG2, and IgG3.
 56. The method of claim 1, wherein thepolypeptide further targets to cells expressing FcRn.
 57. The method ofclaim 1, wherein the polypeptide further binds to one or more FcRn. 58.The method of claim 1, wherein the polypeptide is adapted to treat oneor more disorders selected from the group consisting of the autoimmunedisease and the inflammatory disease.
 59. The method of claim 1, whereinthe polypeptide promotes apoptosis, promotes necrosis, promotes lysis,decreases cell division of neoplastic cells, decreases the number ofvirally infected cells, or a combination thereof.
 60. The method ofclaim 1, wherein the polypeptide is adapted to alter complement binding.61. The method of claim 1, wherein the polypeptide is soluble in aqueoussolution.
 62. The method of claim 1, wherein the polypeptide can targetone or more cells selected from the group consisting of NK cells,monocytes, macrophages, T cells, and B cells.
 63. The method of claim 1,wherein the polypeptide is adapted for use as a vaccine.
 64. The methodof claim 1, wherein the polypeptide is adapted for use as an antigenpresenting vehicle for one or more antigens.
 65. The method of claim 1,wherein the polypeptide is adapted for use as a vehicle for immunologictolerance induction.
 66. The method of claim 1, wherein the secondregion comprises from three to five copies of the at least a portion ofthe HCH2 region.
 67. The method of claim 1, wherein the second regioncomprises an altered hinge region in the HCH2 region, which (a)preserves targeting to cells expressing low affinity FcγR or preservesbinding to low affinity FcγR and (b) prevents inter-molecular disulfidebond formation.
 68. The method of claim 1, wherein the polypeptide isbetween 26 kDa and 1500 kDa.
 69. The method of claim 1, wherein thepolypeptide is between 45 kDa and 600 kDa.
 70. The method of claim 1,wherein the administering comprises administering from about 0.05 mg toabout 10 mg of polypeptide/kg body weight.
 71. The method of claim 1,wherein the protein or portion thereof comprises a growth factor or aportion thereof.
 72. The method of claim 71, wherein the growth factoris an epidermal growth factor.
 73. The method of claim 72, wherein theepidermal growth factor is an erbB-2 protein.
 74. The method of claim35, wherein the tumor antigen sequence comprises an erbB-2 protein orportion thereof.
 75. The method of claim 50, wherein the tumor expressedantigen comprises an erbB-2 protein or portion thereof.
 76. A methodcomprising: administering a polypeptide to at least one cell in vitro,wherein the polypeptide consists of a dimer of a first amino acid chainand a second amino acid chain, which can be the same or different, andwhere each amino acid chain comprises: a first region comprising aprotein or portion thereof; a second region comprising two to six copiesof at least a portion of an HCH2 region of a first mammalian IgG, wherethe HCH2 region comprises a hinge of the first mammalian IgG and a CH2of the first mammalian IgG; and a third region comprising a hinge of asecond mammalian IgG, a CH2 of the second mammalian IgG, and a CH3 ofthe second mammalian IgG; wherein (1) one or more cysteines of the hingeof the HCH2 region of one or both amino acid chains is mutated to anamino acid selected from the group consisting of serine, alanine,glycine, and threonine, (2) the first mammalian IgG and the secondmammalian IgG are from the same mammal, and (3) the polypeptide targetsto cells expressing a low affinity FcγR or binds to a low affinity FcγR.